CA3148714A1 - Tsg6 polypeptide fragment for dry eye disease - Google Patents
Tsg6 polypeptide fragment for dry eye disease Download PDFInfo
- Publication number
- CA3148714A1 CA3148714A1 CA3148714A CA3148714A CA3148714A1 CA 3148714 A1 CA3148714 A1 CA 3148714A1 CA 3148714 A CA3148714 A CA 3148714A CA 3148714 A CA3148714 A CA 3148714A CA 3148714 A1 CA3148714 A1 CA 3148714A1
- Authority
- CA
- Canada
- Prior art keywords
- link
- tsg6
- polypeptide
- dry eye
- tsg6 polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 122
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 120
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 120
- 208000003556 Dry Eye Syndromes Diseases 0.000 title claims abstract description 108
- 239000012634 fragment Substances 0.000 title description 16
- 238000011282 treatment Methods 0.000 claims abstract description 80
- 230000007547 defect Effects 0.000 claims description 47
- 230000004489 tear production Effects 0.000 claims description 47
- 239000000203 mixture Substances 0.000 claims description 44
- 239000003889 eye drop Substances 0.000 claims description 35
- 238000009472 formulation Methods 0.000 claims description 35
- 210000002175 goblet cell Anatomy 0.000 claims description 34
- 206010061218 Inflammation Diseases 0.000 claims description 33
- 210000004087 cornea Anatomy 0.000 claims description 33
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 33
- 230000004054 inflammatory process Effects 0.000 claims description 33
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 claims description 32
- 208000035475 disorder Diseases 0.000 claims description 27
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 102000004127 Cytokines Human genes 0.000 claims description 20
- 108090000695 Cytokines Proteins 0.000 claims description 20
- 102100032807 Tumor necrosis factor-inducible gene 6 protein Human genes 0.000 claims description 17
- 230000000694 effects Effects 0.000 claims description 16
- 230000002265 prevention Effects 0.000 claims description 16
- 108010036949 Cyclosporine Proteins 0.000 claims description 15
- 208000021386 Sjogren Syndrome Diseases 0.000 claims description 15
- 229960001265 ciclosporin Drugs 0.000 claims description 14
- 229930182912 cyclosporin Natural products 0.000 claims description 14
- 230000035876 healing Effects 0.000 claims description 13
- 210000004561 lacrimal apparatus Anatomy 0.000 claims description 12
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 claims description 12
- 229960005205 prednisolone Drugs 0.000 claims description 12
- 229960005381 lifitegrast Drugs 0.000 claims description 10
- JFOZKMSJYSPYLN-QHCPKHFHSA-N lifitegrast Chemical compound CS(=O)(=O)C1=CC=CC(C[C@H](NC(=O)C=2C(=C3CCN(CC3=CC=2Cl)C(=O)C=2C=C3OC=CC3=CC=2)Cl)C(O)=O)=C1 JFOZKMSJYSPYLN-QHCPKHFHSA-N 0.000 claims description 10
- 239000000607 artificial tear Substances 0.000 claims description 9
- 230000001629 suppression Effects 0.000 claims description 9
- 230000000770 proinflammatory effect Effects 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 238000011200 topical administration Methods 0.000 claims description 6
- 108090001005 Interleukin-6 Proteins 0.000 claims description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 5
- 102100040247 Tumor necrosis factor Human genes 0.000 claims description 5
- 101710169430 Tumor necrosis factor-inducible gene 6 protein Proteins 0.000 claims 1
- 230000027455 binding Effects 0.000 description 111
- 238000000034 method Methods 0.000 description 33
- 238000010186 staining Methods 0.000 description 32
- 208000014674 injury Diseases 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 28
- 230000006378 damage Effects 0.000 description 28
- 208000027418 Wounds and injury Diseases 0.000 description 26
- 206010013774 Dry eye Diseases 0.000 description 24
- 230000001965 increasing effect Effects 0.000 description 23
- 238000012360 testing method Methods 0.000 description 21
- 101000847156 Homo sapiens Tumor necrosis factor-inducible gene 6 protein Proteins 0.000 description 19
- 230000002757 inflammatory effect Effects 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 229940053174 restasis Drugs 0.000 description 17
- 208000024891 symptom Diseases 0.000 description 17
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 16
- 239000002953 phosphate buffered saline Substances 0.000 description 16
- KZMRYBLIGYQPPP-UHFFFAOYSA-M 3-[[4-[(2-chlorophenyl)-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]cyclohexa-2,5-dien-1-ylidene]methyl]-n-ethylanilino]methyl]benzenesulfonate Chemical compound C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)Cl)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 KZMRYBLIGYQPPP-UHFFFAOYSA-M 0.000 description 14
- 238000011740 C57BL/6 mouse Methods 0.000 description 14
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 14
- 239000008194 pharmaceutical composition Substances 0.000 description 14
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 14
- 229920002674 hyaluronan Polymers 0.000 description 13
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 230000009841 epithelial lesion Effects 0.000 description 12
- 229940099552 hyaluronan Drugs 0.000 description 12
- 101100028791 Caenorhabditis elegans pbs-5 gene Proteins 0.000 description 11
- 230000009467 reduction Effects 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 231100000673 dose–response relationship Toxicity 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 7
- 206010012601 diabetes mellitus Diseases 0.000 description 7
- 210000003560 epithelium corneal Anatomy 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 6
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 6
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 6
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 6
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 6
- 102100021936 C-C motif chemokine 27 Human genes 0.000 description 6
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 6
- 102100032366 C-C motif chemokine 7 Human genes 0.000 description 6
- 102100025279 C-X-C motif chemokine 11 Human genes 0.000 description 6
- 102100036153 C-X-C motif chemokine 6 Human genes 0.000 description 6
- 229920002971 Heparan sulfate Polymers 0.000 description 6
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 6
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 6
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 6
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 description 6
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 6
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 description 6
- 101000858060 Homo sapiens C-X-C motif chemokine 11 Proteins 0.000 description 6
- 101000947177 Homo sapiens C-X-C motif chemokine 6 Proteins 0.000 description 6
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 6
- 101000582950 Homo sapiens Platelet factor 4 Proteins 0.000 description 6
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 6
- 102100026236 Interleukin-8 Human genes 0.000 description 6
- 102100030304 Platelet factor 4 Human genes 0.000 description 6
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 6
- 229940112869 bone morphogenetic protein Drugs 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 239000006196 drop Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 230000007613 environmental effect Effects 0.000 description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 6
- 238000012744 immunostaining Methods 0.000 description 6
- 238000010172 mouse model Methods 0.000 description 6
- 238000011201 multiple comparisons test Methods 0.000 description 6
- 239000003921 oil Substances 0.000 description 6
- 238000001543 one-way ANOVA Methods 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 206010011026 Corneal lesion Diseases 0.000 description 5
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical group CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 5
- 108010025832 RANK Ligand Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 102100024568 Tumor necrosis factor ligand superfamily member 11 Human genes 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000003995 emulsifying agent Substances 0.000 description 5
- 230000008378 epithelial damage Effects 0.000 description 5
- 210000000981 epithelium Anatomy 0.000 description 5
- 239000003925 fat Substances 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- 210000001165 lymph node Anatomy 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 238000002203 pretreatment Methods 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 230000002269 spontaneous effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000008733 trauma Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 4
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 description 4
- 210000000068 Th17 cell Anatomy 0.000 description 4
- 230000003110 anti-inflammatory effect Effects 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000006071 cream Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 229940012356 eye drops Drugs 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 210000004907 gland Anatomy 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 239000002085 irritant Substances 0.000 description 4
- 231100000021 irritant Toxicity 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000011514 reflex Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- 229930000680 A04AD01 - Scopolamine Natural products 0.000 description 3
- 102100036601 Aggrecan core protein Human genes 0.000 description 3
- 108010067219 Aggrecans Proteins 0.000 description 3
- 229920000045 Dermatan sulfate Polymers 0.000 description 3
- 206010015150 Erythema Diseases 0.000 description 3
- 102100037362 Fibronectin Human genes 0.000 description 3
- 108010067306 Fibronectins Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 101001082142 Homo sapiens Pentraxin-related protein PTX3 Proteins 0.000 description 3
- STECJAGHUSJQJN-GAUPFVANSA-N Hyoscine Natural products C1([C@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-GAUPFVANSA-N 0.000 description 3
- -1 IFNy Proteins 0.000 description 3
- 101800001691 Inter-alpha-trypsin inhibitor light chain Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- STECJAGHUSJQJN-UHFFFAOYSA-N N-Methyl-scopolamin Natural products C1C(C2C3O2)N(C)C3CC1OC(=O)C(CO)C1=CC=CC=C1 STECJAGHUSJQJN-UHFFFAOYSA-N 0.000 description 3
- 102100027351 Pentraxin-related protein PTX3 Human genes 0.000 description 3
- 102100032859 Protein AMBP Human genes 0.000 description 3
- 108010067787 Proteoglycans Proteins 0.000 description 3
- 102000016611 Proteoglycans Human genes 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 108700012920 TNF Proteins 0.000 description 3
- 210000000447 Th1 cell Anatomy 0.000 description 3
- 108010046722 Thrombospondin 1 Proteins 0.000 description 3
- 102100036034 Thrombospondin-1 Human genes 0.000 description 3
- 102000018594 Tumour necrosis factor Human genes 0.000 description 3
- 108050007852 Tumour necrosis factor Proteins 0.000 description 3
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 229940094517 chondroitin 4-sulfate Drugs 0.000 description 3
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 3
- 229940051593 dermatan sulfate Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 102000050255 human TNFAIP6 Human genes 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000002669 organ and tissue protective effect Effects 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000037390 scarring Effects 0.000 description 3
- STECJAGHUSJQJN-FWXGHANASA-N scopolamine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-FWXGHANASA-N 0.000 description 3
- 229960002646 scopolamine Drugs 0.000 description 3
- 238000012800 visualization Methods 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 2
- 108010049976 Bone Morphogenetic Protein 5 Proteins 0.000 description 2
- 108010049974 Bone Morphogenetic Protein 6 Proteins 0.000 description 2
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 2
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 2
- 102100022526 Bone morphogenetic protein 5 Human genes 0.000 description 2
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 description 2
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 2
- 208000009043 Chemical Burns Diseases 0.000 description 2
- 208000018380 Chemical injury Diseases 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 208000028006 Corneal injury Diseases 0.000 description 2
- 206010055665 Corneal neovascularisation Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000010412 Glaucoma Diseases 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- 102100035379 Growth/differentiation factor 5 Human genes 0.000 description 2
- 101710204282 Growth/differentiation factor 5 Proteins 0.000 description 2
- 102100035368 Growth/differentiation factor 6 Human genes 0.000 description 2
- 101710204281 Growth/differentiation factor 6 Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 2
- 208000009319 Keratoconjunctivitis Sicca Diseases 0.000 description 2
- 102000010954 Link domains Human genes 0.000 description 2
- 108050001157 Link domains Proteins 0.000 description 2
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 2
- 206010065062 Meibomian gland dysfunction Diseases 0.000 description 2
- 208000022873 Ocular disease Diseases 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 231100000168 Stevens-Johnson syndrome Toxicity 0.000 description 2
- 102100029529 Thrombospondin-2 Human genes 0.000 description 2
- 206010044223 Toxic epidermal necrolysis Diseases 0.000 description 2
- 231100000087 Toxic epidermal necrolysis Toxicity 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- 206010047513 Vision blurred Diseases 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 210000002159 anterior chamber Anatomy 0.000 description 2
- 229940072107 ascorbate Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 238000011260 co-administration Methods 0.000 description 2
- 210000000795 conjunctiva Anatomy 0.000 description 2
- 201000000159 corneal neovascularization Diseases 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000001804 debridement Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000000744 eyelid Anatomy 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 235000012701 green S Nutrition 0.000 description 2
- WDPIZEKLJKBSOZ-UHFFFAOYSA-M green s Chemical compound [Na+].C1=CC(N(C)C)=CC=C1C(C=1C2=CC=C(C=C2C=C(C=1O)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=CC(=[N+](C)C)C=C1 WDPIZEKLJKBSOZ-UHFFFAOYSA-M 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 230000000508 neurotrophic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000003883 ointment base Substances 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920002704 polyhistidine Polymers 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- HLZKNKRTKFSKGZ-UHFFFAOYSA-N tetradecan-1-ol Chemical compound CCCCCCCCCCCCCCO HLZKNKRTKFSKGZ-UHFFFAOYSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 108010060887 thrombospondin 2 Proteins 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- QMMJWQMCMRUYTG-UHFFFAOYSA-N 1,2,4,5-tetrachloro-3-(trifluoromethyl)benzene Chemical compound FC(F)(F)C1=C(Cl)C(Cl)=CC(Cl)=C1Cl QMMJWQMCMRUYTG-UHFFFAOYSA-N 0.000 description 1
- LGEZTMRIZWCDLW-UHFFFAOYSA-N 14-methylpentadecyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCCCCCCCCCCCCCC(C)C LGEZTMRIZWCDLW-UHFFFAOYSA-N 0.000 description 1
- SFAAOBGYWOUHLU-UHFFFAOYSA-N 2-ethylhexyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(CC)CCCC SFAAOBGYWOUHLU-UHFFFAOYSA-N 0.000 description 1
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000010470 Ageusia Diseases 0.000 description 1
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 206010006784 Burning sensation Diseases 0.000 description 1
- 239000004358 Butane-1, 3-diol Substances 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 206010051625 Conjunctival hyperaemia Diseases 0.000 description 1
- 208000006069 Corneal Opacity Diseases 0.000 description 1
- 206010051559 Corneal defect Diseases 0.000 description 1
- 206010011013 Corneal erosion Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 206010066786 Diabetic keratopathy Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102100034719 Extracellular glycoprotein lacritin Human genes 0.000 description 1
- 208000020564 Eye injury Diseases 0.000 description 1
- 208000035620 Eye penetration Diseases 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101001090521 Homo sapiens Extracellular glycoprotein lacritin Proteins 0.000 description 1
- 101000998146 Homo sapiens Interleukin-17A Proteins 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102100033461 Interleukin-17A Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 206010023642 Lacrimation decreased Diseases 0.000 description 1
- 206010023644 Lacrimation increased Diseases 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 102000019298 Lipocalin Human genes 0.000 description 1
- 108050006654 Lipocalin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- GCKMFJBGXUYNAG-HLXURNFRSA-N Methyltestosterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)CC2 GCKMFJBGXUYNAG-HLXURNFRSA-N 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 208000023715 Ocular surface disease Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 208000015323 Penetrating Eye injury Diseases 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241001282135 Poromitra oscitans Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 206010037508 Punctate keratitis Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 1
- 102100030511 Stanniocalcin-1 Human genes 0.000 description 1
- 101710142157 Stanniocalcin-1 Proteins 0.000 description 1
- 108010062276 T-Cell Acute Lymphocytic Leukemia Protein 1 Proteins 0.000 description 1
- 102100040365 T-cell acute lymphocytic leukemia protein 1 Human genes 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 206010064996 Ulcerative keratitis Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 208000010011 Vitamin A Deficiency Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 206010048232 Yawning Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000005299 abrasion Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000019666 ageusia Nutrition 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229940125715 antihistaminic agent Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000004397 blinking Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000008355 cartilage degradation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 229940082500 cetostearyl alcohol Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 231100000269 corneal opacity Toxicity 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 201000004400 dacryoadenitis Diseases 0.000 description 1
- SASYSVUEVMOWPL-NXVVXOECSA-N decyl oleate Chemical compound CCCCCCCCCCOC(=O)CCCCCCC\C=C/CCCCCCCC SASYSVUEVMOWPL-NXVVXOECSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 231100000223 dermal penetration Toxicity 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000008387 emulsifying waxe Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000002657 hormone replacement therapy Methods 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000008309 hydrophilic cream Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000006749 inflammatory damage Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 231100001032 irritation of the eye Toxicity 0.000 description 1
- 229940078545 isocetyl stearate Drugs 0.000 description 1
- XUGNVMKQXJXZCD-UHFFFAOYSA-N isopropyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)C XUGNVMKQXJXZCD-UHFFFAOYSA-N 0.000 description 1
- 206010023365 keratopathy Diseases 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 230000004317 lacrimation Effects 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 208000018769 loss of vision Diseases 0.000 description 1
- 231100000864 loss of vision Toxicity 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229940043348 myristyl alcohol Drugs 0.000 description 1
- 210000002850 nasal mucosa Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000001640 nerve ending Anatomy 0.000 description 1
- 206010069732 neurotrophic keratopathy Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000004273 ophthalmic nerve Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000008251 pharmaceutical emulsion Substances 0.000 description 1
- IMACFCSSMIZSPP-UHFFFAOYSA-N phenacyl chloride Chemical compound ClCC(=O)C1=CC=CC=C1 IMACFCSSMIZSPP-UHFFFAOYSA-N 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 230000001012 protector Effects 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012755 real-time RT-PCR analysis Methods 0.000 description 1
- 201000002002 recurrent corneal erosion Diseases 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229930187593 rose bengal Natural products 0.000 description 1
- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 description 1
- 229940081623 rose bengal Drugs 0.000 description 1
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000008137 solubility enhancer Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 208000024205 superior limbic keratoconjunctivitis Diseases 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000004488 tear evaporation Effects 0.000 description 1
- 239000003491 tear gas Substances 0.000 description 1
- 229940085503 testred Drugs 0.000 description 1
- OULAJFUGPPVRBK-UHFFFAOYSA-N tetratriacontyl alcohol Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCO OULAJFUGPPVRBK-UHFFFAOYSA-N 0.000 description 1
- 229960004523 tiletamine Drugs 0.000 description 1
- 239000008181 tonicity modifier Substances 0.000 description 1
- 239000012049 topical pharmaceutical composition Substances 0.000 description 1
- 102000042565 transient receptor (TC 1.A.4) family Human genes 0.000 description 1
- 108091053409 transient receptor (TC 1.A.4) family Proteins 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000004034 viscosity adjusting agent Substances 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4718—Cytokine-induced proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
- A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
- A61K38/13—Cyclosporins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/04—Artificial tears; Irrigation solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/08—Vasodilators for multiple indications
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Ophthalmology & Optometry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Marine Sciences & Fisheries (AREA)
- Transplantation (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the treatment of dry eye disease and particularly, although not exclusively, to the treatment of dry eye disease with a LINK_TSG6 polypeptide.
Description
2 PCT/EP2020/067448 This application claims priority from GB1910645.9 filed 25 July 2019, the contents and elements of which are herein incorporated by reference for all purposes.
Field of the Invention The present invention relates to the treatment of ocular surface disorders and particularly, although not exclusively, to the treatment of dry eye disease with a LINK_TSG6 polypeptide.
Background Tumour necrosis factor (TNF)-stimulated gene 6 (TSG-6) is a ¨35 kDa secreted product of TNF-stimulated gene-6, expressed in response to inflammatory mediators and growth factors.
While constitutively expressed in a few tissues, TSG-6 is generally upregulated wherever there is inflammation. For the most part TSG-6 exhibits anti-inflammatory and tissue protective properties, but has been implicated as sometimes playing a role in disease pathology, for example, in the lung. While being made by a broad range of cell types, it was the finding that TSG-6 is produced by mesenchymal stem/stromal cells (MSCs) in response to inflammatory signals, and that it mediates many of their immunomodulatory and reparative activities, which has led to a wealth of publications on the therapeutic effects of this intriguing molecule across a wide range of disease models.
TSG-6 is a relatively small protein, with a molecular mass of only ¨35-38 kDa, being mainly composed of two modular domains. Given TSG-6's size, it has a surprisingly large number of activities, including the modulation of immune and stromal cell function and its contribution to extracellular matrix formation, mechanics and remodelling. It is the ability of TSG-6 to regulate matrix organization, and to control the association of matrix molecules with cell surface receptors and with extracellular signalling factors (e.g.
chemokines), that likely underlies its diverse functional repertoire. In this regard, TSG-6 interacts with a large array of ligands, such as glycosaminoglycans (GAGs), proteoglycan (PG) core proteins and other matrix components, and binds directly to multiple chemokines and bone morphogenetic proteins (BMPs).
One particularly unusual function of TSG-6 is its role as an enzyme that catalyses the covalent modification of the non-sulfated GAG hyaluronan (HA) with so-called heavy chains (HCs) from the inter-a-inhibitor (Id) family of proteoglycans. This process, mediated by the full-length TSG6 protein, but not LINK_TSG6 polypeptides containing only a fragment of TSG-6, results in the formation of HC.HA
complexes, and is essential for mammalian ovulation and fertilisation, and also occurs in many other contexts (e.g. inflammation) where HC=HAs either confer tissue protection or contribute to pathological processes.
The sites and contexts of TSG-6 expression, its structure and ligand-binding properties, and how these together underpin its diverse biology and therapeutic potential at a molecular level are reviewed in Day &
Milner (Matrix Biology (2019) 78-79, 60-83).
US2015/0057229 describes the use of LINK_TSG6 in inhibiting cartilage degradation.
Dry eye disease (also known as keratoconjunctivitis sicca) is one of the most common ocular diseases, occurring in between 7% and 33% of the population worldwide. It is a multifactorial condition of the tear film and ocular surface and is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface, neurotrophic deficiency and meibomian gland dysfunction.
Kim etal. (2016) (Cornea 35(4), 536-542) compared topically applied TSG-6, cyclosporine and prednisolone for treating dry eye. 12 week old NOD.B10.H2b mice were topically administered with recombinant TSG-6 (0.1%) 4 times a day, 0.05% cyclosporine (Restasis) twice a day, or 1% prednisolone (Pred Forte) 4 times a day for 1 week. Topical TSG-6 was found to be as effective in inflammation mediated dry eye as cyclosporine eye drops. However, they conclude that clinical application of TSG-6 is limited by various factors, including difficulty in large-scale production or variation in stability of the recombinant protein.
W02011/139357 described the use of adult stem cells/progenitor cells and stem cell proteins for the treatment of eye injuries and diseases. They propose therapy based on the discovery that after a chemical burn to the cornea of a rat, application of MSCs or MSC conditioned medium reduced inflammation and revascularisation. They proposed the use of anti-apoptotic and anti-inflammatory proteins such as STC-1 and TSG-6, which are expressed by mesenchymal stem cells. Corneal surface inflammation was created in rat eyes by ethanol application and mechanical debridement of the corneal and limbal epithelium. Application of recombinant full length TSG-6 resulted in reduced corneal opacity and neovascularization as compared to a PBS control, and the authors conclude that proteins produced by MSCs in response to an injury signal can protect the corneal surface from damage by increasing the viability and proliferation of corneal epithelial progenitors and by suppressing inflammation at the corneal surface.
U52016/0075750 (Prockop et al) described a method of producing a protein or polypeptide, such as TSG-6 protein, in mammalian cells suspended in a protein-free medium that includes at least one agent that suppresses production of hyaluronic acid, hyaluronan or a salt thereof.
The present invention has been devised in light of the above considerations.
Summary of the Invention The present disclosure relates to LINK_TSG6 polypeptide for use in the treatment or prevention of ocular surface disorder such as dry eye disease or other ocular surface disorders with corneal lesions similar to those observed in dry eye disease. The inventors have found that this polypeptide is capable of reducing or preventing the signs and symptoms of dry eye disease, in a dose-dependent manner. Surprisingly, the
Field of the Invention The present invention relates to the treatment of ocular surface disorders and particularly, although not exclusively, to the treatment of dry eye disease with a LINK_TSG6 polypeptide.
Background Tumour necrosis factor (TNF)-stimulated gene 6 (TSG-6) is a ¨35 kDa secreted product of TNF-stimulated gene-6, expressed in response to inflammatory mediators and growth factors.
While constitutively expressed in a few tissues, TSG-6 is generally upregulated wherever there is inflammation. For the most part TSG-6 exhibits anti-inflammatory and tissue protective properties, but has been implicated as sometimes playing a role in disease pathology, for example, in the lung. While being made by a broad range of cell types, it was the finding that TSG-6 is produced by mesenchymal stem/stromal cells (MSCs) in response to inflammatory signals, and that it mediates many of their immunomodulatory and reparative activities, which has led to a wealth of publications on the therapeutic effects of this intriguing molecule across a wide range of disease models.
TSG-6 is a relatively small protein, with a molecular mass of only ¨35-38 kDa, being mainly composed of two modular domains. Given TSG-6's size, it has a surprisingly large number of activities, including the modulation of immune and stromal cell function and its contribution to extracellular matrix formation, mechanics and remodelling. It is the ability of TSG-6 to regulate matrix organization, and to control the association of matrix molecules with cell surface receptors and with extracellular signalling factors (e.g.
chemokines), that likely underlies its diverse functional repertoire. In this regard, TSG-6 interacts with a large array of ligands, such as glycosaminoglycans (GAGs), proteoglycan (PG) core proteins and other matrix components, and binds directly to multiple chemokines and bone morphogenetic proteins (BMPs).
One particularly unusual function of TSG-6 is its role as an enzyme that catalyses the covalent modification of the non-sulfated GAG hyaluronan (HA) with so-called heavy chains (HCs) from the inter-a-inhibitor (Id) family of proteoglycans. This process, mediated by the full-length TSG6 protein, but not LINK_TSG6 polypeptides containing only a fragment of TSG-6, results in the formation of HC.HA
complexes, and is essential for mammalian ovulation and fertilisation, and also occurs in many other contexts (e.g. inflammation) where HC=HAs either confer tissue protection or contribute to pathological processes.
The sites and contexts of TSG-6 expression, its structure and ligand-binding properties, and how these together underpin its diverse biology and therapeutic potential at a molecular level are reviewed in Day &
Milner (Matrix Biology (2019) 78-79, 60-83).
US2015/0057229 describes the use of LINK_TSG6 in inhibiting cartilage degradation.
Dry eye disease (also known as keratoconjunctivitis sicca) is one of the most common ocular diseases, occurring in between 7% and 33% of the population worldwide. It is a multifactorial condition of the tear film and ocular surface and is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface, neurotrophic deficiency and meibomian gland dysfunction.
Kim etal. (2016) (Cornea 35(4), 536-542) compared topically applied TSG-6, cyclosporine and prednisolone for treating dry eye. 12 week old NOD.B10.H2b mice were topically administered with recombinant TSG-6 (0.1%) 4 times a day, 0.05% cyclosporine (Restasis) twice a day, or 1% prednisolone (Pred Forte) 4 times a day for 1 week. Topical TSG-6 was found to be as effective in inflammation mediated dry eye as cyclosporine eye drops. However, they conclude that clinical application of TSG-6 is limited by various factors, including difficulty in large-scale production or variation in stability of the recombinant protein.
W02011/139357 described the use of adult stem cells/progenitor cells and stem cell proteins for the treatment of eye injuries and diseases. They propose therapy based on the discovery that after a chemical burn to the cornea of a rat, application of MSCs or MSC conditioned medium reduced inflammation and revascularisation. They proposed the use of anti-apoptotic and anti-inflammatory proteins such as STC-1 and TSG-6, which are expressed by mesenchymal stem cells. Corneal surface inflammation was created in rat eyes by ethanol application and mechanical debridement of the corneal and limbal epithelium. Application of recombinant full length TSG-6 resulted in reduced corneal opacity and neovascularization as compared to a PBS control, and the authors conclude that proteins produced by MSCs in response to an injury signal can protect the corneal surface from damage by increasing the viability and proliferation of corneal epithelial progenitors and by suppressing inflammation at the corneal surface.
U52016/0075750 (Prockop et al) described a method of producing a protein or polypeptide, such as TSG-6 protein, in mammalian cells suspended in a protein-free medium that includes at least one agent that suppresses production of hyaluronic acid, hyaluronan or a salt thereof.
The present invention has been devised in light of the above considerations.
Summary of the Invention The present disclosure relates to LINK_TSG6 polypeptide for use in the treatment or prevention of ocular surface disorder such as dry eye disease or other ocular surface disorders with corneal lesions similar to those observed in dry eye disease. The inventors have found that this polypeptide is capable of reducing or preventing the signs and symptoms of dry eye disease, in a dose-dependent manner. Surprisingly, the
3 inventors have found that this polypeptide is more potent at reducing corneal epithelial defects than full length TSG-6.
In some cases, the treatment or prevention of dry eye disease described herein comprises one or more effects selected from the group consisting: healing of corneal epithelial defects; increase in tear production; suppression of inflammation; and an increase in, or the preservation of, the number of conjunctival goblet cells. Suppression of inflammation may comprise a decrease in the production of one or more pro-inflammatory cytokines in the cornea, the intraorbital lacrimal glands, or both the cornea and the intraorbital lacrimal glands, the pro-inflammatory cytokines optionally selected from TNFa, IFNy, IL-6 and IL-113.
The treatment or prevention of dry eye disease may comprise healing of corneal epithelial defects, increase in tear production, suppression of inflammation or an increase in, or the preservation of the number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells prior to the administration of LINK_TSG6 polypeptide, and as compared to administration of PBS vehicle.
The treatment or prevention of dry eye disease may comprise healing of corneal epithelial defects, increase in tear production, suppression of inflammation or an increase in, or the preservation of the number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells in a control individual treated with full-length TSG-6 protein.
In some aspects described herein, the treatment comprises topical administration of LINK_TSG6 polypeptide to the eye. The LINK_TSG6 polypeptide may be formulated as an eye drop. The treatment may comprise the administration of an eye drop comprising LINK_TSG6. The LINK_TSG6 polypeptide may be formulated with, or the treatment may involve co-administration with prednisolone, cyclosporine, Lifitegrast (XiidraTm), artificial tears, or any combination thereof.
In some aspects, the treatment comprises administering LINK_TSG6 polypeptide two times per day. In some aspects, the treatment comprises administering LINK_TSG6 polypeptide more than two times per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide fewer than 4 times per day, or fewer than 3 times per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide once per day. In some cases, the treatment comprises administering .. LINK_TSG6 polypeptide less frequently than once per day, such as once every two days, one time every three days, once every week, or once every two weeks.
The treatment may involve the administration of between 10-200 pg LINK_TSG6 per eye, such as between 100 and 200 pg LINK_TSG6 per eye, between 100 and 150 pg LINK_TSG6 per eye, between 120 and 150 pg LINK_TSG6 per eye. Preferably, the treatment involves the administration of around 120pg-150pg LINK_TSG6 polypeptide per eye, or around 12-15 pg LINK_TSG6 per eye.
The treatment or prevention of ocular surface disorders such as dry eye disease disclosed herein is applicable to dry eye disease associated with any cause. The individual being treated may have a
In some cases, the treatment or prevention of dry eye disease described herein comprises one or more effects selected from the group consisting: healing of corneal epithelial defects; increase in tear production; suppression of inflammation; and an increase in, or the preservation of, the number of conjunctival goblet cells. Suppression of inflammation may comprise a decrease in the production of one or more pro-inflammatory cytokines in the cornea, the intraorbital lacrimal glands, or both the cornea and the intraorbital lacrimal glands, the pro-inflammatory cytokines optionally selected from TNFa, IFNy, IL-6 and IL-113.
The treatment or prevention of dry eye disease may comprise healing of corneal epithelial defects, increase in tear production, suppression of inflammation or an increase in, or the preservation of the number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells prior to the administration of LINK_TSG6 polypeptide, and as compared to administration of PBS vehicle.
The treatment or prevention of dry eye disease may comprise healing of corneal epithelial defects, increase in tear production, suppression of inflammation or an increase in, or the preservation of the number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells in a control individual treated with full-length TSG-6 protein.
In some aspects described herein, the treatment comprises topical administration of LINK_TSG6 polypeptide to the eye. The LINK_TSG6 polypeptide may be formulated as an eye drop. The treatment may comprise the administration of an eye drop comprising LINK_TSG6. The LINK_TSG6 polypeptide may be formulated with, or the treatment may involve co-administration with prednisolone, cyclosporine, Lifitegrast (XiidraTm), artificial tears, or any combination thereof.
In some aspects, the treatment comprises administering LINK_TSG6 polypeptide two times per day. In some aspects, the treatment comprises administering LINK_TSG6 polypeptide more than two times per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide fewer than 4 times per day, or fewer than 3 times per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide once per day. In some cases, the treatment comprises administering .. LINK_TSG6 polypeptide less frequently than once per day, such as once every two days, one time every three days, once every week, or once every two weeks.
The treatment may involve the administration of between 10-200 pg LINK_TSG6 per eye, such as between 100 and 200 pg LINK_TSG6 per eye, between 100 and 150 pg LINK_TSG6 per eye, between 120 and 150 pg LINK_TSG6 per eye. Preferably, the treatment involves the administration of around 120pg-150pg LINK_TSG6 polypeptide per eye, or around 12-15 pg LINK_TSG6 per eye.
The treatment or prevention of ocular surface disorders such as dry eye disease disclosed herein is applicable to dry eye disease associated with any cause. The individual being treated may have a
4 condition that is associated with an increased incidence of dry eye disease, such as Sjogren's syndrome, rheumatoid arthritis or diabetes. In some cases, the individual has Type 1 diabetes or Type 2 diabetes.
The individual may be at risk of developing an ocular surface disorder such as dry eye disease due to aging, exposure to air pollution and/or increased use of a visual device (e.g.
smart phone, computer, tablet).
The treatment may involve the administration of LINK_TSG6 polypeptide that comprises, consist, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID
NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ
ID NO: 7 or 9.
Also disclosed herein are methods of treatment or prevention of ocular surface disorders such as dry eye disease. The method may involve the administration of a therapeutically effective amount of LINK_TSG6 to a patient in need thereof. It may involve the topical administration of LINK_TSG6 to the eye.
Another aspect disclosed herein is the use of a LINK_TSG6 polypeptide in the manufacture of a medicament for the treatment or prevention of ocular surface disorders such as dry eye disease. The medicament may be formulated for topical administration to the eye, such as an eye drop.
.. A further aspect disclosed herein is a pharmaceutical composition comprising LINK_TSG6 polypeptide.
The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in saline. The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline. In some aspects, the pharmaceutical composition comprises at least 2000 pg/ml, 2100 pg/ml, 2200 pg/ml, 2300 pg/ml 2400pg/ml, 2500 pg/ml, 2600 pg/ml, 2700 pg/ml, 2800 pg/ml, 2900 pg/ml, 3000 .. pg/ml, 3100 pg/ml, 3200 pg/ml, 3300 pg/ml or more than 3300 pg/ml LINK_TSG6. In some aspects, the pharmaceutical composition comprises at least 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 600 pg/ml, 700 pg/ml, 800 pg/ml, 900 pg/ml, 1000 pg/ml, 1200 pg/ml, 1300 pg/ml, 1400 pg/ml, 1500 pg/ml, 1600 pg/ml, 1700 pg/ml, 1800 pg/ml 1900 pg/ml, 2000 pg/ml LINK_TSG6. In some aspects, the pharmaceutical composition comprises at least 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 600 pg/ml, .. 700 pg/ml, 800 pg/ml, 900 pg/ml, 1000 pg/ml, 1200 pg/ml, 1300 pg/ml, 1400 pg/ml, 1500 pg/ml, 1600 pg/ml, 1700 pg/ml, 1800 pg/ml 1900 pg/ml, 2000 pg/ml LINK_TSG6. Preferably, the pharmaceutical formation comprises at least 2000 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 120 pg and around 150pg of LINK_TSG6 per drop.
The pharmaceutical composition may be an eye drop formulation. The eye drop formulation may comprise between 1500 pg/ml and 3500 pg/ml, between 1500 pg/ml and 3000 pg/ml, between 2000 pg/ml and 3000 pg/ml, or between 2400 pg/ml and 3000 pg/ml. Preferably, the eye drop formulation comprises between about 2400 pg/ml and about 3000 pg/ml. In some cases, the eye drop formulation comprises at least 2200, at least 2300 pg/ml, at least 2400 pg/ml, at least 2500 pg/ml, at least 2600 pg/ml, at least 2700 pg/ml, at least 2800 pg/ml, at least 2900 pg/ml, at least 3000 pg/ml or more than .. 3000 pg/ml. The eye drop formulation may further comprise prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears. The eye drop formulation may comprise a pharmaceutically acceptable carrier. The eye drop formulation may comprise LINK_TSG6 polypeptide solubilised in saline. The eye drop formulation may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline.
The individual may be at risk of developing an ocular surface disorder such as dry eye disease due to aging, exposure to air pollution and/or increased use of a visual device (e.g.
smart phone, computer, tablet).
The treatment may involve the administration of LINK_TSG6 polypeptide that comprises, consist, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID
NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ
ID NO: 7 or 9.
Also disclosed herein are methods of treatment or prevention of ocular surface disorders such as dry eye disease. The method may involve the administration of a therapeutically effective amount of LINK_TSG6 to a patient in need thereof. It may involve the topical administration of LINK_TSG6 to the eye.
Another aspect disclosed herein is the use of a LINK_TSG6 polypeptide in the manufacture of a medicament for the treatment or prevention of ocular surface disorders such as dry eye disease. The medicament may be formulated for topical administration to the eye, such as an eye drop.
.. A further aspect disclosed herein is a pharmaceutical composition comprising LINK_TSG6 polypeptide.
The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in saline. The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline. In some aspects, the pharmaceutical composition comprises at least 2000 pg/ml, 2100 pg/ml, 2200 pg/ml, 2300 pg/ml 2400pg/ml, 2500 pg/ml, 2600 pg/ml, 2700 pg/ml, 2800 pg/ml, 2900 pg/ml, 3000 .. pg/ml, 3100 pg/ml, 3200 pg/ml, 3300 pg/ml or more than 3300 pg/ml LINK_TSG6. In some aspects, the pharmaceutical composition comprises at least 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 600 pg/ml, 700 pg/ml, 800 pg/ml, 900 pg/ml, 1000 pg/ml, 1200 pg/ml, 1300 pg/ml, 1400 pg/ml, 1500 pg/ml, 1600 pg/ml, 1700 pg/ml, 1800 pg/ml 1900 pg/ml, 2000 pg/ml LINK_TSG6. In some aspects, the pharmaceutical composition comprises at least 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 600 pg/ml, .. 700 pg/ml, 800 pg/ml, 900 pg/ml, 1000 pg/ml, 1200 pg/ml, 1300 pg/ml, 1400 pg/ml, 1500 pg/ml, 1600 pg/ml, 1700 pg/ml, 1800 pg/ml 1900 pg/ml, 2000 pg/ml LINK_TSG6. Preferably, the pharmaceutical formation comprises at least 2000 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 120 pg and around 150pg of LINK_TSG6 per drop.
The pharmaceutical composition may be an eye drop formulation. The eye drop formulation may comprise between 1500 pg/ml and 3500 pg/ml, between 1500 pg/ml and 3000 pg/ml, between 2000 pg/ml and 3000 pg/ml, or between 2400 pg/ml and 3000 pg/ml. Preferably, the eye drop formulation comprises between about 2400 pg/ml and about 3000 pg/ml. In some cases, the eye drop formulation comprises at least 2200, at least 2300 pg/ml, at least 2400 pg/ml, at least 2500 pg/ml, at least 2600 pg/ml, at least 2700 pg/ml, at least 2800 pg/ml, at least 2900 pg/ml, at least 3000 pg/ml or more than .. 3000 pg/ml. The eye drop formulation may further comprise prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears. The eye drop formulation may comprise a pharmaceutically acceptable carrier. The eye drop formulation may comprise LINK_TSG6 polypeptide solubilised in saline. The eye drop formulation may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline.
5 The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
Summary of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
Figure 1. Sequences relevant to the present disclosure.
Figure 2. LINK_TSG6 reduces the signs of dry eye disease. A. Ocular staining score following lissamine green staining; B. Aqueous tear production as determined by phenol red thread test; C. Pro-inflammatory cytokine mRNA level as determined by real-time RT-PCR; D. Conjunctival goblet cell counts in PAS
(Periodic acid¨Schiff stain) stained conjunctival sections. For A and B
significance was determined by Wilcoxon matched-pairs signed rank test for comparison between pre- and post-treatment and Mann-Whitney U test for comparison between PBS and LINK_TSG6. ns = not significant, p>0.05; ** = p<0.01;
***= p<0.001. For C and D significance was determined by one-way ANOVA and Tukey's multiple comparisons test. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01; ***=
p<0.001; ****= p<0.0001.
Figure 3. LINK_TSG6 reduces the signs of dry eye disease in a dose-dependent manner. A.
Quantitation of corneal epithelial defects from lissamine green-stained corneas. LINK_TSG6 1 pg and 0.1 pg was effective in reducing corneal epithelial defects, but LINK_TSG6 0.01 pg did not significantly improve corneal epithelial defects. B. Quantification of aqueous tear production by phenol red thread test.
LINK_TSG6 1 pg and 0.1 pg were both effective in increasing the amount of tear production, but LINK_TSG6 0.01 pg did not significantly improve tear production. C.
Quantification of pro-inflammatory cytokines by real-time RT-PCR analysis. LINK_TSG6 1 pg was most effective in suppressing TNF-a expression. In A and B significance was determined by Wilcoxon matched-pairs signed rank test. * =
p<0.05; ** = p<0.01; ns = not significant, p>0.05. In C significance was determined by one-way ANOVA
and Tukey's multiple comparisons test. ns = not significant, p>0.05; *=
p<0.05; ** = p<0.01 ***= p<0.001;
****= p<0.0001.
Figure 4. Dose response data for LINK_TSG6. A. Quantitation of corneal epithelial defects from lissamine green-stained corneas by ocular staining; B. Quantification of aqueous tear production by phenol red thread test; C. Pro-inflammatory cytokine mRNA level as determined by real-time RT-PCR D.
Conjunctival goblet cell counts in PAS stained conjunctival sections.
Significance determined by one-way ANOVA and Tukey's multiple comparisons tests. ns = not significant, p>0.05; *=
p<0.05; ** = p<0.01 ***
= p<0.001; **** = p<0.0001.
Figure 5. LINK_TSG6 is more effective than full length human recombinant TSG-6 (FL TSG6) at reducing the signs of dry eye disease. A. Ocular staining score following lissamine green staining; B. Aqueous tear production as determined by phenol red thread test; C. Conjunctival goblet cell counts in PAS stained conjunctival sections D. Histological analysis of CD3 immunostaining in lacrimal gland as expressed by the number of foci with CD3 cell infiltration. Equivalent molar doses are compared (92, 9.2 and 0.92
Summary of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
Figure 1. Sequences relevant to the present disclosure.
Figure 2. LINK_TSG6 reduces the signs of dry eye disease. A. Ocular staining score following lissamine green staining; B. Aqueous tear production as determined by phenol red thread test; C. Pro-inflammatory cytokine mRNA level as determined by real-time RT-PCR; D. Conjunctival goblet cell counts in PAS
(Periodic acid¨Schiff stain) stained conjunctival sections. For A and B
significance was determined by Wilcoxon matched-pairs signed rank test for comparison between pre- and post-treatment and Mann-Whitney U test for comparison between PBS and LINK_TSG6. ns = not significant, p>0.05; ** = p<0.01;
***= p<0.001. For C and D significance was determined by one-way ANOVA and Tukey's multiple comparisons test. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01; ***=
p<0.001; ****= p<0.0001.
Figure 3. LINK_TSG6 reduces the signs of dry eye disease in a dose-dependent manner. A.
Quantitation of corneal epithelial defects from lissamine green-stained corneas. LINK_TSG6 1 pg and 0.1 pg was effective in reducing corneal epithelial defects, but LINK_TSG6 0.01 pg did not significantly improve corneal epithelial defects. B. Quantification of aqueous tear production by phenol red thread test.
LINK_TSG6 1 pg and 0.1 pg were both effective in increasing the amount of tear production, but LINK_TSG6 0.01 pg did not significantly improve tear production. C.
Quantification of pro-inflammatory cytokines by real-time RT-PCR analysis. LINK_TSG6 1 pg was most effective in suppressing TNF-a expression. In A and B significance was determined by Wilcoxon matched-pairs signed rank test. * =
p<0.05; ** = p<0.01; ns = not significant, p>0.05. In C significance was determined by one-way ANOVA
and Tukey's multiple comparisons test. ns = not significant, p>0.05; *=
p<0.05; ** = p<0.01 ***= p<0.001;
****= p<0.0001.
Figure 4. Dose response data for LINK_TSG6. A. Quantitation of corneal epithelial defects from lissamine green-stained corneas by ocular staining; B. Quantification of aqueous tear production by phenol red thread test; C. Pro-inflammatory cytokine mRNA level as determined by real-time RT-PCR D.
Conjunctival goblet cell counts in PAS stained conjunctival sections.
Significance determined by one-way ANOVA and Tukey's multiple comparisons tests. ns = not significant, p>0.05; *=
p<0.05; ** = p<0.01 ***
= p<0.001; **** = p<0.0001.
Figure 5. LINK_TSG6 is more effective than full length human recombinant TSG-6 (FL TSG6) at reducing the signs of dry eye disease. A. Ocular staining score following lissamine green staining; B. Aqueous tear production as determined by phenol red thread test; C. Conjunctival goblet cell counts in PAS stained conjunctival sections D. Histological analysis of CD3 immunostaining in lacrimal gland as expressed by the number of foci with CD3 cell infiltration. Equivalent molar doses are compared (92, 9.2 and 0.92
6 pmol) corresponding to 1, 0.1 and 0.01 g LINK_TSG6 and 3.27, 0.327 and 0.0327 g FL TSG-6, respectively. Significance determined by one-way ANOVA and Tukey's multiple comparisons test. * =
p<0.05.
Figure 6. Evaluation of LINK_TSG6 in a desiccation injury-induced dry eye model and comparison with Restasis. A. Ocular staining prior to the induction of desiccation injury and pre-treatment; B. Ocular staining post-desiccation injury and post-treatment; C. Tear production prior to the induction of desiccation injury and pre-treatment; D. Tear production post-desiccation injury and post-treatment; E.
Th1 cells in draining cervical lymph nodes; F. Th17 cells in draining cervical lymph nodes. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01 ***= p<0.001; ****= p<0.0001.
Figure 7. Evaluation of LINK_TSG6 in already-desiccated mice. A. Ocular staining; B. Tear production;
C. Conjunctival goblet cell counts in PAS stained conjunctival sections following treatment; D. MMP-9 mRNA levels at the ocular surface; ns = not significant, p>0.05; *= p<0.05; **
= p<0.01 ***= p<0.001; ****
= p<0.0001. A&B Wilcoxon matched-pairs signed rank test for comparison between pre- and post-treatment. C&D one-way ANOVA and Tukey's multiple comparisons test.
Figure 8. Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A.
Ocular staining score following lissamine green staining, prior to treatment; B. Ocular staining score following lissamine green staining following treatment; C. Quantification of aqueous tear production by phenol red thread test pre-treatment D. Quantification of aqueous tear production by phenol red thread test post-treatment E.
Conjunctival goblet cell counts in PAS stained conjunctival sections following treatment. F. Histological analysis of CD3 immunostaining in lacrimal gland. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01 ***
= p<0.001; ****= p<0.0001, one-way ANOVA and Tukey's multiple comparisons test.
Detailed Description of the Invention Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
The present invention provides a method for treating or preventing ocular surface disorders such as dry eye disease, which method comprises administering to a subject a LINK_TSG6 polypeptide. The inventors have shown that LINK_TSG6 is more potent than recombinant human TSG-6 in reducing corneal epithelial lesions and treating or reducing the signs or symptoms of ocular surface disorders such as dry eye disease. Without wishing to be bound by theory, this may be a result of improved penetration into the tissue (due to the smaller size of the molecule), differences in the biodistribution as compared to the full length protein, or the absence of signalling or enzymatic activities effected by the CUB_C domain.
In this regard, full length TSG-6 binds more effectively to HA than LINK_TSG6 since the former interaction is cooperative (and likely involves CUB_C domain) (Baranova et al., 2011 J. Biol. Chem. 286, 25675-25686). HA has been implicated in some anti-inflammatory activities (see Day and Milner 2019).
p<0.05.
Figure 6. Evaluation of LINK_TSG6 in a desiccation injury-induced dry eye model and comparison with Restasis. A. Ocular staining prior to the induction of desiccation injury and pre-treatment; B. Ocular staining post-desiccation injury and post-treatment; C. Tear production prior to the induction of desiccation injury and pre-treatment; D. Tear production post-desiccation injury and post-treatment; E.
Th1 cells in draining cervical lymph nodes; F. Th17 cells in draining cervical lymph nodes. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01 ***= p<0.001; ****= p<0.0001.
Figure 7. Evaluation of LINK_TSG6 in already-desiccated mice. A. Ocular staining; B. Tear production;
C. Conjunctival goblet cell counts in PAS stained conjunctival sections following treatment; D. MMP-9 mRNA levels at the ocular surface; ns = not significant, p>0.05; *= p<0.05; **
= p<0.01 ***= p<0.001; ****
= p<0.0001. A&B Wilcoxon matched-pairs signed rank test for comparison between pre- and post-treatment. C&D one-way ANOVA and Tukey's multiple comparisons test.
Figure 8. Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A.
Ocular staining score following lissamine green staining, prior to treatment; B. Ocular staining score following lissamine green staining following treatment; C. Quantification of aqueous tear production by phenol red thread test pre-treatment D. Quantification of aqueous tear production by phenol red thread test post-treatment E.
Conjunctival goblet cell counts in PAS stained conjunctival sections following treatment. F. Histological analysis of CD3 immunostaining in lacrimal gland. ns = not significant, p>0.05; *= p<0.05; ** = p<0.01 ***
= p<0.001; ****= p<0.0001, one-way ANOVA and Tukey's multiple comparisons test.
Detailed Description of the Invention Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
The present invention provides a method for treating or preventing ocular surface disorders such as dry eye disease, which method comprises administering to a subject a LINK_TSG6 polypeptide. The inventors have shown that LINK_TSG6 is more potent than recombinant human TSG-6 in reducing corneal epithelial lesions and treating or reducing the signs or symptoms of ocular surface disorders such as dry eye disease. Without wishing to be bound by theory, this may be a result of improved penetration into the tissue (due to the smaller size of the molecule), differences in the biodistribution as compared to the full length protein, or the absence of signalling or enzymatic activities effected by the CUB_C domain.
In this regard, full length TSG-6 binds more effectively to HA than LINK_TSG6 since the former interaction is cooperative (and likely involves CUB_C domain) (Baranova et al., 2011 J. Biol. Chem. 286, 25675-25686). HA has been implicated in some anti-inflammatory activities (see Day and Milner 2019).
7 TSG-6 (Tumor Necrosis Factor-Stimulated Gene-6) TSG-6 is a secreted protein composed of two modular domains. TSG-6 is not usually constitutively expressed in adult tissues, rather being induced in response to inflammatory mediators. During inflammation, TSG-6 is an endogenous protector of tissues. Many of the immunomodulatory and tissue-protective effects of MSCs are mediated by their secretion of TSG-6.
Recombinant full-length TSG-6 protein has been shown to have anti-inflammatory and tissue protective effects in a wide range of disease models, such as atherosclerosis, myocardial infarction, hypertrophic scarring, colitis, autoimmune diabetes, rheumatoid arthritis, traumatic brain injury or acute lung injury.
Full length TSG-6 is hard to make, insoluble and prone to aggregation. As disclosed herein, these disadvantages are not associated with LINK_TSG6, a short recombinant peptide comprising the LINK
module of human TSG-6. This short polypeptide is easier to make than full length TSG6, and is highly soluble and stable in solution.
LINK_TSG6 polypeptide as disclosed herein comprises only the Link module of human or mammalian TSG-6. In some embodiments, the TSG-6 polypeptide comprises or consists essentially of the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 5. The Link module corresponds to residues 37-128 of SEQ ID NO:s 2 and 5, and is shown in SEQ ID NO: 7. In some preferred aspects, the LINK_TSG6 polypeptides useful in the present invention do not comprise some or all of residues 1-35 of the full length TSG6 sequence of SEQ ID NO: 2 or 5 at the N terminal, The Link module is responsible for the hyaluronan (HA) binding activity, chondroitin-4-sulfate binding activity, aggrecan binding activity, inter-a-inhibitor (Id) binding activity, bikunin binding activity, versican binding activity, dermatan sulfate binding activity, pentraxin-3 binding activity, thrombospondin-1 binding activity, thrombospondin-2 binding activity, fibronectin binding activity, heparin/heparan sulfate binding activity, RANKL binding activity of TSG-6, bone morphogenetic protein (BMP) -2 binding activity, BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
LINK_TSG6 may be a fragment of TSG-6 exhibiting one or more of hyaluronan (HA) binding activity, chondroitin-4-sulfate binding activity, aggrecan binding activity, inter-a-inhibitor (Id) binding activity, bikunin binding activity, versican binding activity, dermatan sulfate binding activity, pentraxin-3 binding activity, thrombospondin-1 binding activity, thrombospondin-2 binding activity, fibronectin binding activity, heparin/heparan sulfate binding activity, RANKL binding activity, bone morphogenetic protein (BMP) -2 binding activity, BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity.
Recombinant full-length TSG-6 protein has been shown to have anti-inflammatory and tissue protective effects in a wide range of disease models, such as atherosclerosis, myocardial infarction, hypertrophic scarring, colitis, autoimmune diabetes, rheumatoid arthritis, traumatic brain injury or acute lung injury.
Full length TSG-6 is hard to make, insoluble and prone to aggregation. As disclosed herein, these disadvantages are not associated with LINK_TSG6, a short recombinant peptide comprising the LINK
module of human TSG-6. This short polypeptide is easier to make than full length TSG6, and is highly soluble and stable in solution.
LINK_TSG6 polypeptide as disclosed herein comprises only the Link module of human or mammalian TSG-6. In some embodiments, the TSG-6 polypeptide comprises or consists essentially of the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 5. The Link module corresponds to residues 37-128 of SEQ ID NO:s 2 and 5, and is shown in SEQ ID NO: 7. In some preferred aspects, the LINK_TSG6 polypeptides useful in the present invention do not comprise some or all of residues 1-35 of the full length TSG6 sequence of SEQ ID NO: 2 or 5 at the N terminal, The Link module is responsible for the hyaluronan (HA) binding activity, chondroitin-4-sulfate binding activity, aggrecan binding activity, inter-a-inhibitor (Id) binding activity, bikunin binding activity, versican binding activity, dermatan sulfate binding activity, pentraxin-3 binding activity, thrombospondin-1 binding activity, thrombospondin-2 binding activity, fibronectin binding activity, heparin/heparan sulfate binding activity, RANKL binding activity of TSG-6, bone morphogenetic protein (BMP) -2 binding activity, BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
LINK_TSG6 may be a fragment of TSG-6 exhibiting one or more of hyaluronan (HA) binding activity, chondroitin-4-sulfate binding activity, aggrecan binding activity, inter-a-inhibitor (Id) binding activity, bikunin binding activity, versican binding activity, dermatan sulfate binding activity, pentraxin-3 binding activity, thrombospondin-1 binding activity, thrombospondin-2 binding activity, fibronectin binding activity, heparin/heparan sulfate binding activity, RANKL binding activity, bone morphogenetic protein (BMP) -2 binding activity, BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity.
8 The LINK domain of TSG-6 (LINK_TSG6) may be the region of full-length TSG-6 N-terminal to the CUB_C domain. As such, the LINK_TSG6 protein may lack all or part of the CUB_C
domain.
The LINK domain may contain the amino acid sequence of SEQ ID NO: 7 or SEQ ID
NO: 9. LINK_TSG6 polypeptide comprises, consists, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9.
LINK_TSG6 is preferably a polypeptide comprising or consisting of: (i) the amino acid sequence of SEQ
ID NO: 7 or 9, or (ii) an amino acid sequence having one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7 or 9.
Accordingly, the LINK_TSG6 polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 7;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 7 and having RANKL binding activity; or (c) a fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
The LINK_TSG6 polypeptide may consists of, or consist essentially of, the sequence shown in SEQ ID
NO: 7.
SEQ ID NO: 9 shows a recombinant polypeptide which includes the Link module of TSG-6 (LINK_TSG6).
Accordingly, the TSG-6 polypeptide used in the invention may preferably comprises:
(a) the amino acid sequence of SEQ ID NO: 9;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 9 and having RANKL binding activity; or (c) a fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity,CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
The LINK_TSG6 polypeptide preferably consists of, or consists essentially of, the sequence shown in SEQ ID NO: 9.
In some aspects described herein, the LINK_TSG6 polypeptide is not conjugated to an active agent, such as an antibody or antigen binding fragment. For example, in some cases, the LINK_TSG6 polypeptide is not conjugated to an IL-17A antibody or fragment thereof. In some cases, the LINK_TSG6 polypeptide comprises or consists of a single copy of LINK_TSG6 polypeptide, such as a single copy of SEQ ID NO:
7 or SEQ ID NO:9. In other words, in these cases, the LINK_TSG6 polypeptide does not comprise a plurality of LINK module sequences, such as a plurality of copies of SEQ ID
NO: 7 or SEQ ID NO: 9. In some cases, the LINK_TSG6 polypeptide does not comprise a His tag, such as a 6xHIS tag.
domain.
The LINK domain may contain the amino acid sequence of SEQ ID NO: 7 or SEQ ID
NO: 9. LINK_TSG6 polypeptide comprises, consists, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9.
LINK_TSG6 is preferably a polypeptide comprising or consisting of: (i) the amino acid sequence of SEQ
ID NO: 7 or 9, or (ii) an amino acid sequence having one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7 or 9.
Accordingly, the LINK_TSG6 polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 7;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 7 and having RANKL binding activity; or (c) a fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
The LINK_TSG6 polypeptide may consists of, or consist essentially of, the sequence shown in SEQ ID
NO: 7.
SEQ ID NO: 9 shows a recombinant polypeptide which includes the Link module of TSG-6 (LINK_TSG6).
Accordingly, the TSG-6 polypeptide used in the invention may preferably comprises:
(a) the amino acid sequence of SEQ ID NO: 9;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 9 and having RANKL binding activity; or (c) a fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity,CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity..
The LINK_TSG6 polypeptide preferably consists of, or consists essentially of, the sequence shown in SEQ ID NO: 9.
In some aspects described herein, the LINK_TSG6 polypeptide is not conjugated to an active agent, such as an antibody or antigen binding fragment. For example, in some cases, the LINK_TSG6 polypeptide is not conjugated to an IL-17A antibody or fragment thereof. In some cases, the LINK_TSG6 polypeptide comprises or consists of a single copy of LINK_TSG6 polypeptide, such as a single copy of SEQ ID NO:
7 or SEQ ID NO:9. In other words, in these cases, the LINK_TSG6 polypeptide does not comprise a plurality of LINK module sequences, such as a plurality of copies of SEQ ID
NO: 7 or SEQ ID NO: 9. In some cases, the LINK_TSG6 polypeptide does not comprise a His tag, such as a 6xHIS tag.
9 Amino acid identity may be calculated using any suitable algorithm. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux etal. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST
algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul (1993) J. MoL EvoL 36, 290-300; Altschul etal. (1990) J. Mol. Biol. 215, 403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul etal., supra).
These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.
The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X
determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (VV) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA
89, 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The variant sequences typically differ by at least 1, 2, 5, 10, 20, 30, 50 or more mutations (which can be substitutions, deletions or insertions of amino acids). For example, from 1 to 50, 2 to 30, 3 to 20 or 5 to 10 amino acid substitutions, deletions or insertions can be made. The modified polypeptide may generally retain CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity, preferably in a dose-dependent manner. The substitutions are preferably conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul (1993) J. MoL EvoL 36, 290-300; Altschul etal. (1990) J. Mol. Biol. 215, 403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul etal., supra).
These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.
The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X
determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (VV) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA
89, 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The variant sequences typically differ by at least 1, 2, 5, 10, 20, 30, 50 or more mutations (which can be substitutions, deletions or insertions of amino acids). For example, from 1 to 50, 2 to 30, 3 to 20 or 5 to 10 amino acid substitutions, deletions or insertions can be made. The modified polypeptide may generally retain CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity, preferably in a dose-dependent manner. The substitutions are preferably conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
10 I L V
Polar - uncharged CSTM
NQ
Polar - charged D E
KR
AROMATIC H F WY
A LINK_TSG6 polypeptide used in the invention is typically at least 10, for example at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more amino acids in length, up to 100, 150, 200 or 250 amino acids in length, as long as it retains the CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity of TSG-6. Preferably, the polypeptide includes the sequence shown in SEQ ID NO: 7. Fragments of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID
NO: 9 preferably contain the residues shown to be essential for hyaluronan binding in Mahoney etal. (2001) J. Biol. Chem.
276, 22764-22771 and Blundell etal. (2003) J. Biol. Chem. 278, 49261-49270.
Fragments of the amino acid sequence of SEQ ID NO: 2 or 5 preferably contain the residues Lys-46 and/or Tyr-47 and/or Tyr-94 and/or Phe-105 and/or Tyr-113 of SEQ ID NO: 2 or 5. Most preferably, the fragment of SEQ ID NO: 2 or 5 contains each of residues Lys-46, Tyr- 47, Tyr-94, Phe-105 and Tyr-113 of SEQ
ID NO: 2 0r5.
Fragments of the amino acid sequence of SEQ ID NO: 7 may be used in the invention. Such fragments preferably contain the residues Lys-10 and/or Tyr-11 and/or Tyr-58 and/or Phe-69 and/or Tyr-77 of SEQ
ID NO: 7. Most preferably, the fragment of SEQ ID NO: 7 contains each of residues Lys-10, Tyr-11, Tyr-58, Phe-69 and Tyr-77 of SEQ ID NO: 7.
Fragments of the amino acid sequence of SEQ ID NO: 9 preferably contain the residues Lys-11 and/or Tyr-12 and/or Tyr-59 and/or Phe-70 and/or Tyr-78 of SEQ ID NO: 9. Most preferably, the fragment of SEQ ID NO: 9 contains each of residues Lys-11, Tyr-12, Tyr-59, Phe-70 and Tyr-78 of SEQ ID NO: 9.
The TSG-6 polypeptides used in the invention may be chemically modified, e.g.
post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues. They may be modified by the addition of histidine residues to assist their purification or by the addition of a transmembrane sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term "polypeptide" used herein.
Suitable assays for determining the ability of a TSG-6 polypeptide to bind to HA, chondroitin-4-sulfate, aggrecan, inter-a-inhibitor (lap, bikunin, versican, dermatan sulfate, pentraxin-3, thrombospondin-1, heparin/heparan sulfate, fibronectin and RANKL are well-known in the art (Getting etal. (2002) J. Biol.
Chem. 277, 51068-51076; Mahoney et al. (2005) J. Biol. Chem. 280, 27044-27055;
Salustri et al. (2004) .. Development 131, 1577-1586; Parkar et al. (1997) FEBS Lett. 410, 413-417;
Parkar et aL (1998) FEBS
Lett. 428, 171-176; Mahoney etal. (2001) J. Biol. Chem. 276, 22764-22771;
Nentwich etal. (2002) J. Biol Chem. 211, 15354-15362; Kuznetsova etal. (2005) J. Biol. Chem. 280, 30899-30908), Dyer etal. (2014)
Polar - uncharged CSTM
NQ
Polar - charged D E
KR
AROMATIC H F WY
A LINK_TSG6 polypeptide used in the invention is typically at least 10, for example at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more amino acids in length, up to 100, 150, 200 or 250 amino acids in length, as long as it retains the CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity of TSG-6. Preferably, the polypeptide includes the sequence shown in SEQ ID NO: 7. Fragments of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID
NO: 9 preferably contain the residues shown to be essential for hyaluronan binding in Mahoney etal. (2001) J. Biol. Chem.
276, 22764-22771 and Blundell etal. (2003) J. Biol. Chem. 278, 49261-49270.
Fragments of the amino acid sequence of SEQ ID NO: 2 or 5 preferably contain the residues Lys-46 and/or Tyr-47 and/or Tyr-94 and/or Phe-105 and/or Tyr-113 of SEQ ID NO: 2 or 5. Most preferably, the fragment of SEQ ID NO: 2 or 5 contains each of residues Lys-46, Tyr- 47, Tyr-94, Phe-105 and Tyr-113 of SEQ
ID NO: 2 0r5.
Fragments of the amino acid sequence of SEQ ID NO: 7 may be used in the invention. Such fragments preferably contain the residues Lys-10 and/or Tyr-11 and/or Tyr-58 and/or Phe-69 and/or Tyr-77 of SEQ
ID NO: 7. Most preferably, the fragment of SEQ ID NO: 7 contains each of residues Lys-10, Tyr-11, Tyr-58, Phe-69 and Tyr-77 of SEQ ID NO: 7.
Fragments of the amino acid sequence of SEQ ID NO: 9 preferably contain the residues Lys-11 and/or Tyr-12 and/or Tyr-59 and/or Phe-70 and/or Tyr-78 of SEQ ID NO: 9. Most preferably, the fragment of SEQ ID NO: 9 contains each of residues Lys-11, Tyr-12, Tyr-59, Phe-70 and Tyr-78 of SEQ ID NO: 9.
The TSG-6 polypeptides used in the invention may be chemically modified, e.g.
post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues. They may be modified by the addition of histidine residues to assist their purification or by the addition of a transmembrane sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term "polypeptide" used herein.
Suitable assays for determining the ability of a TSG-6 polypeptide to bind to HA, chondroitin-4-sulfate, aggrecan, inter-a-inhibitor (lap, bikunin, versican, dermatan sulfate, pentraxin-3, thrombospondin-1, heparin/heparan sulfate, fibronectin and RANKL are well-known in the art (Getting etal. (2002) J. Biol.
Chem. 277, 51068-51076; Mahoney et al. (2005) J. Biol. Chem. 280, 27044-27055;
Salustri et al. (2004) .. Development 131, 1577-1586; Parkar et al. (1997) FEBS Lett. 410, 413-417;
Parkar et aL (1998) FEBS
Lett. 428, 171-176; Mahoney etal. (2001) J. Biol. Chem. 276, 22764-22771;
Nentwich etal. (2002) J. Biol Chem. 211, 15354-15362; Kuznetsova etal. (2005) J. Biol. Chem. 280, 30899-30908), Dyer etal. (2014)
11 J. Immunol 192, 2177-2185; Dyer etal. (2016) J. Biol. Chem. 291, 12627-12640;
and Mahoney etal.
(2008) J. Biol. Chem 283, 25952-25962.
TSG-6 polypeptides for use in the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A
polypeptide for use in the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
LINK_TSG6 polypeptides for use in the present invention may be natural or non-naturally occurring polypeptides. Polypeptides may be isolated from any suitable organism that expresses a TSG-6 polypeptide. The TSG-6 polypeptide may be isolated from a human or another suitable mammal, such as primates, rats or mice. Alternatively, TSG-6 polypeptide may be isolated from a fish or an amphibian.
Polypeptides for use in the invention may also be prepared as fragments of such isolated polypeptides.
Further, the LINK_TSG6 polypeptides may also be made synthetically or by recombinant means. For example, a recombinant LINK_TSG6 polypeptide may be produced by transfecting cells in culture with an expression vector comprising a nucleotide sequence encoding the polypeptide operably linked to suitable control sequences, culturing the cells, extracting and purifying the LINK_TSG6 polypeptide produced by the cells. Methods for the recombinant production of polypeptides are well-known in the art (for example, Sambrook etal., 2001 , Molecular Cloning: a laboratory manual, 31d edition, Cold Harbour Laboratory Press). Preferably, the LINK_TSG6 polypeptide is made in a bacteria, such as E. coli. Preferably, the LINK_TSG6 polypeptide is not made in a CHO cell or other mammalian cell. As will be appreciated by those skilled in the art, proteins and polypeptides made in a bacteria such as E. coli will completely or substantially lack glycosylation, whereas glycosylation is a common feature of proteins and polypeptides made in a mammalian cell such as a CHO cell. In particular, the LINK_TSG6 according to the present disclosure may lack N-linked glycosylation on Asn118 (SEQ ID NO: 2 or SEQ ID
NO: 5).
The amino acid sequence of LINK_TSG6 polypeptides for use in the invention may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the polypeptides are produced by synthetic means, such amino acids may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production. In some aspects, the LINK_TSG6 polypeptides described herein do not comprise a polyhistidine tag, such as a 6His tag. In some aspects, the LINK_TSG6 polypeptides described herein do not comprise a polyhistidine tag at the C-terminal of the polypeptide.
LINK_TSG6 polypeptides for use in the invention may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N
orientation. This is conventional in the art for producing such polypeptides.
A number of side chain modifications are known in the art and may be made to the side chains of the LINK_TSG6 polypeptides, provided that the polypeptides retain corneal defect healing activity.
and Mahoney etal.
(2008) J. Biol. Chem 283, 25952-25962.
TSG-6 polypeptides for use in the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A
polypeptide for use in the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
LINK_TSG6 polypeptides for use in the present invention may be natural or non-naturally occurring polypeptides. Polypeptides may be isolated from any suitable organism that expresses a TSG-6 polypeptide. The TSG-6 polypeptide may be isolated from a human or another suitable mammal, such as primates, rats or mice. Alternatively, TSG-6 polypeptide may be isolated from a fish or an amphibian.
Polypeptides for use in the invention may also be prepared as fragments of such isolated polypeptides.
Further, the LINK_TSG6 polypeptides may also be made synthetically or by recombinant means. For example, a recombinant LINK_TSG6 polypeptide may be produced by transfecting cells in culture with an expression vector comprising a nucleotide sequence encoding the polypeptide operably linked to suitable control sequences, culturing the cells, extracting and purifying the LINK_TSG6 polypeptide produced by the cells. Methods for the recombinant production of polypeptides are well-known in the art (for example, Sambrook etal., 2001 , Molecular Cloning: a laboratory manual, 31d edition, Cold Harbour Laboratory Press). Preferably, the LINK_TSG6 polypeptide is made in a bacteria, such as E. coli. Preferably, the LINK_TSG6 polypeptide is not made in a CHO cell or other mammalian cell. As will be appreciated by those skilled in the art, proteins and polypeptides made in a bacteria such as E. coli will completely or substantially lack glycosylation, whereas glycosylation is a common feature of proteins and polypeptides made in a mammalian cell such as a CHO cell. In particular, the LINK_TSG6 according to the present disclosure may lack N-linked glycosylation on Asn118 (SEQ ID NO: 2 or SEQ ID
NO: 5).
The amino acid sequence of LINK_TSG6 polypeptides for use in the invention may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the polypeptides are produced by synthetic means, such amino acids may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production. In some aspects, the LINK_TSG6 polypeptides described herein do not comprise a polyhistidine tag, such as a 6His tag. In some aspects, the LINK_TSG6 polypeptides described herein do not comprise a polyhistidine tag at the C-terminal of the polypeptide.
LINK_TSG6 polypeptides for use in the invention may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N
orientation. This is conventional in the art for producing such polypeptides.
A number of side chain modifications are known in the art and may be made to the side chains of the LINK_TSG6 polypeptides, provided that the polypeptides retain corneal defect healing activity.
12 Ocular Surface Disorders The present disclosure relates to the treatment or prevention of ocular surface disorders. Ocular surface disorders include Dry Eye Disease (DED), persistent corneal epitheliopathy (non-healing epithelial defects) associated with diabetic keratopathy, neurotrophic keratopathy, exposure keratopathy or limbal deficiency; contact lens-/eye drop-induced epithelial erosions; ocular graft versus host disease (GVHD);
Stevens-Johnson syndrome (SJS); toxic epidermal necrolysis (TEN), ocular surface dysfunction in glaucoma patients, corneal wounds resulting from glaucoma surgery recurrent corneal erosion, superficial punctate keratitis, superior limbic keratoconjunctivitis.
Dry Eye Disease The present disclosure relates to the treatment or prevention of dry eye disease. Dry eye disease (also known as dry eye syndrome, or keratoconjunctivitis sicca) is the condition of having dry eyes, and affects 7-33% of the world population. Dry eye disease occurs when not enough tears are produced (aqueous deficient dry eye disease), or when tears evaporate too quickly (evaporative dry eye disease). Causes include aging (which is known to cause lacrimal grand atrophy and inflammation), infection, exposure to environmental irritants such as smoke, contact lens use, meibomian gland dysfunction, allergies, pregnancy, Sjogren's syndrome, vitamin A deficiency, laser eye treatment, or as a result of medication such as antihistamines, blood pressure medication, hormone replacement therapy and some antidepressants. In some case, dry eye disease occurs from activities that are associated with a reduced blinking rate, such as the use of screens such as computer monitors, smart phones or tablets, televisions or driving. Dry eye disease may result in tiny abrasions on the surface of the eyes (i.e. defects in corneal epithelium. Dry eye disease may result in pathologic changes in the corneal epithelium, such as squamous metaplasia and loss of goblet cells, in severe cases leading to corneal erosion, ulceration, neovascularization and scarring, or thinning and perforation. A diagnosis of dry eye disease may involve standardized dry eye questionnaire (The Ocular Surface Disease Index, or SDI), a 12-item scale for the assessment of symptoms related to dry eye disease and their effect on vision.
Dry eye disease is a disease resulting from desiccation injury to the ocular surface, or inflammatory damage to the lacrimal gland. It is clinically distinct from other trauma induced ocular disorders such as chemical burn or trauma resulting from direct exposure of the ocular surface to chemicals (such as alcohol), physical or chemical debridement, blunt force trauma to the eye, penetrating eye injury, or other ocular wounding, although these trauma induced ocular conditions may result in the subsequent development of dry eye disease.
Signs and symptoms of dry eye disease include irritation, redness, discharge, easily fatigued eyes and blurred vision. The signs and symptoms range from mild and occasional to severe and continuous, and scarring of the cornea may result if left untreated.
Stevens-Johnson syndrome (SJS); toxic epidermal necrolysis (TEN), ocular surface dysfunction in glaucoma patients, corneal wounds resulting from glaucoma surgery recurrent corneal erosion, superficial punctate keratitis, superior limbic keratoconjunctivitis.
Dry Eye Disease The present disclosure relates to the treatment or prevention of dry eye disease. Dry eye disease (also known as dry eye syndrome, or keratoconjunctivitis sicca) is the condition of having dry eyes, and affects 7-33% of the world population. Dry eye disease occurs when not enough tears are produced (aqueous deficient dry eye disease), or when tears evaporate too quickly (evaporative dry eye disease). Causes include aging (which is known to cause lacrimal grand atrophy and inflammation), infection, exposure to environmental irritants such as smoke, contact lens use, meibomian gland dysfunction, allergies, pregnancy, Sjogren's syndrome, vitamin A deficiency, laser eye treatment, or as a result of medication such as antihistamines, blood pressure medication, hormone replacement therapy and some antidepressants. In some case, dry eye disease occurs from activities that are associated with a reduced blinking rate, such as the use of screens such as computer monitors, smart phones or tablets, televisions or driving. Dry eye disease may result in tiny abrasions on the surface of the eyes (i.e. defects in corneal epithelium. Dry eye disease may result in pathologic changes in the corneal epithelium, such as squamous metaplasia and loss of goblet cells, in severe cases leading to corneal erosion, ulceration, neovascularization and scarring, or thinning and perforation. A diagnosis of dry eye disease may involve standardized dry eye questionnaire (The Ocular Surface Disease Index, or SDI), a 12-item scale for the assessment of symptoms related to dry eye disease and their effect on vision.
Dry eye disease is a disease resulting from desiccation injury to the ocular surface, or inflammatory damage to the lacrimal gland. It is clinically distinct from other trauma induced ocular disorders such as chemical burn or trauma resulting from direct exposure of the ocular surface to chemicals (such as alcohol), physical or chemical debridement, blunt force trauma to the eye, penetrating eye injury, or other ocular wounding, although these trauma induced ocular conditions may result in the subsequent development of dry eye disease.
Signs and symptoms of dry eye disease include irritation, redness, discharge, easily fatigued eyes and blurred vision. The signs and symptoms range from mild and occasional to severe and continuous, and scarring of the cornea may result if left untreated.
13 As disclosed herein, the peptide LINK_TSG6 may be used to treat or prevent dry eye disease.
Administration of LINK_TSG6 may result in a healing of corneal epithelial defects, an increase in tear production, the suppression of inflammation and/or an increase/preservation in the number of conjunctival goblet cells. The healing of corneal epithelial defects may result in a reduction in the number of corneal epithelial defects or the size of corneal epithelial defects. Preferably, the healing of corneal epithelial defects results in a reduction in the proportion of the corneal surface that comprises corneal epithelial defects.
Corneal epithelial defects are areas of epithelial (outermost corneal layer) loss, and may be due to mechanical trauma, corneal dryness, neurotrophic cornea, post surgical changes or any other of a variety of etiologies. LINK_TSG6 may be used to reduce or repair corneal epithelial defects. The presence or absence of corneal epithelial defects may be determined by scoring. Corneal epithelial defects may be visualised by staining. For example, through the use of lissamine green or fluorescein dye. The stain is applied to the cornea, and the area of epithelial defect is stained, thereby allowing visualisation of a defect.
An increase in tear production may be determined by Schirmer's test or phenol red thread test.
The number of goblet cell in the conjunctiva may be determined by impression cytology. A cellulose acetate filter paper is applied with pressure to the conjunctival surface for collection of superficial layer and subjected to PAS staining to stain mucin-secreting goblet cells. The number of goblet cells is calculated in the PAS-stained slide.
The presence, absence or amount of inflammation at the ocular surface may be determined by observation of conjunctival redness or conjunctival epithelial defects.
Inflammation may cause disruption of corneal and conjunctival epithelium.
The level of inflammatory cytokines may be determined in a sample of tear from the patient. Tear samples may be obtained using Schirmer Tear Test strips. Nucleic acid and/or protein may be extracted from the tear strip by incubating the strip in ammonium bicarbonate and acetone. The level of inflammatory cytokines may be quantified or qualified by RT-PCR or ELISA. The inflammatory cytokines may be selected from IFN-y, TNF-a, IL-113 and IL-6. In some cases, the inflammatory cytokines may be quantified or qualified by immunoassay. In some cases, the level of MMP9 is determined in a semi-quantitative manner (positive, trace, or negative) using an "InflammaDry" kit in human eyes.
The cornea is the transparent front part of the eye that covers the iris, pupil and the anterior chamber.
The cornea, with the anterior chamber and lens, refracts light, with the cornea accounting for approximately two-thirds of the eye's total optical power. While the cornea contributes to most of the eye's focusing power, its focus is fixed. The cornea has unmyelinated nerve endings sensitive to touch, temperature and chemicals; a touch of the cornea causes an involuntary reflex to close the eyelid.
Because transparency is of prime importance, the healthy cornea does not have or need blood vessels within it. Instead, oxygen dissolves in tears and then diffuses throughout the cornea to keep it healthy.
Similarly, nutrients are transported via diffusion from the tear fluid through the outside surface and the
Administration of LINK_TSG6 may result in a healing of corneal epithelial defects, an increase in tear production, the suppression of inflammation and/or an increase/preservation in the number of conjunctival goblet cells. The healing of corneal epithelial defects may result in a reduction in the number of corneal epithelial defects or the size of corneal epithelial defects. Preferably, the healing of corneal epithelial defects results in a reduction in the proportion of the corneal surface that comprises corneal epithelial defects.
Corneal epithelial defects are areas of epithelial (outermost corneal layer) loss, and may be due to mechanical trauma, corneal dryness, neurotrophic cornea, post surgical changes or any other of a variety of etiologies. LINK_TSG6 may be used to reduce or repair corneal epithelial defects. The presence or absence of corneal epithelial defects may be determined by scoring. Corneal epithelial defects may be visualised by staining. For example, through the use of lissamine green or fluorescein dye. The stain is applied to the cornea, and the area of epithelial defect is stained, thereby allowing visualisation of a defect.
An increase in tear production may be determined by Schirmer's test or phenol red thread test.
The number of goblet cell in the conjunctiva may be determined by impression cytology. A cellulose acetate filter paper is applied with pressure to the conjunctival surface for collection of superficial layer and subjected to PAS staining to stain mucin-secreting goblet cells. The number of goblet cells is calculated in the PAS-stained slide.
The presence, absence or amount of inflammation at the ocular surface may be determined by observation of conjunctival redness or conjunctival epithelial defects.
Inflammation may cause disruption of corneal and conjunctival epithelium.
The level of inflammatory cytokines may be determined in a sample of tear from the patient. Tear samples may be obtained using Schirmer Tear Test strips. Nucleic acid and/or protein may be extracted from the tear strip by incubating the strip in ammonium bicarbonate and acetone. The level of inflammatory cytokines may be quantified or qualified by RT-PCR or ELISA. The inflammatory cytokines may be selected from IFN-y, TNF-a, IL-113 and IL-6. In some cases, the inflammatory cytokines may be quantified or qualified by immunoassay. In some cases, the level of MMP9 is determined in a semi-quantitative manner (positive, trace, or negative) using an "InflammaDry" kit in human eyes.
The cornea is the transparent front part of the eye that covers the iris, pupil and the anterior chamber.
The cornea, with the anterior chamber and lens, refracts light, with the cornea accounting for approximately two-thirds of the eye's total optical power. While the cornea contributes to most of the eye's focusing power, its focus is fixed. The cornea has unmyelinated nerve endings sensitive to touch, temperature and chemicals; a touch of the cornea causes an involuntary reflex to close the eyelid.
Because transparency is of prime importance, the healthy cornea does not have or need blood vessels within it. Instead, oxygen dissolves in tears and then diffuses throughout the cornea to keep it healthy.
Similarly, nutrients are transported via diffusion from the tear fluid through the outside surface and the
14 aqueous humour through the inside surface. Nutrients also come via neurotrophins supplied by the nerves of the cornea. In humans, the cornea has a diameter of about 11.5 mm and a thickness of 0.5-0.6 mm in the center and 0.6-0.8 mm at the periphery. Transparency, avascularity, the presence of immature resident immune cells, and immunologic privilege makes the cornea a very special tissue.
The methods disclosed herein are particularly concerned with the corneal epithelium, and damage thereto. The corneal epithelium is an exceedingly thin (approximately 50 pm) multicellular epithelial tissue layer (non-keratinized stratified squamous epithelium) of fast-growing and easily regenerated cells, kept moist with tears. The corneal epithelium is made up of epithelial cells and covers the front of the cornea. It acts as a frontline barrier to protect the cornea, resisting the free flow of fluids from the tears, and prevents bacteria from entering the cornea and inside of the eye.
Irregularity or defects of the corneal epithelium disrupts the smoothness of the air/tear-film interface, the most significant component of the total refractive power of the eye, thereby reducing visual acuity. It is continuous with the conjunctival epithelium, and is composed of about 6 layers of cells which are shed constantly on the exposed layer and are regenerated by multiplication in the basal layer. In dry eye disease, the corneal epithelium often becomes damaged.
In some methods, tear production is increased, or the thickness of the tear film coating the eye is increased. In some cases, tear retention is decreased, such that tears are evaporated quickly from the eye. Tearing (also known as lacrimation or lachrymation) is the reflex secretion of tears in response to external or internal irritants. Tears are a bodily fluid which may serve to clean and lubricate the eyes in response to irritation. In healthy mammalian eyes, the cornea is continually kept wet and nourished by basal tears. They lubricate the eye, and help to keep it clear of dust. Tear fluid contains water, mucin, lipids, lysozyme, lactoferrin, lipocalin, lacritin, immunoglobulins, glucose, urea, sodium, and potassium.
Some of the substances in lacrimal fluid (such as lysozyme) fight against bacterial infection as a part of the immune system. Lysozyme does this by dissolving a layer in the outer coating, called peptidoglycan, of certain bacteria. Tears are a typical body fluid with a salt content similar to blood plasma. Usually, in a 24-hour period, 0.75 to 1.1 grams (0.03-0.04 ounce avoirdupois) of tears is secreted; this rate slows with age. In addition, the basal tears are composed of antioxidants such as ascorbate, urate, cysteine, glutathione, and tyrosine. Ascorbate and urate constitute half of the tears.
A second type of tears results from irritation of the eye by foreign particles, or from the presence of irritant substances such as onion vapors, perfumes and other fragrances, tear gas, or pepper spray in the eye's environment, including the cornea, conjunctiva, or nasal mucosa, which trigger TRP channels in the ophthalmic nerve. It can also occur with bright light and hot or peppery stimuli to the tongue and mouth. It is also linked with vomiting, coughing and yawning. These reflex tears attempt to wash out irritants that may have come into contact with the eye.
The methods disclosed herein may relate to preservation or improvement of tear production, such as basal tears or reflex tears.
The methods disclosed herein are particularly concerned with the corneal epithelium, and damage thereto. The corneal epithelium is an exceedingly thin (approximately 50 pm) multicellular epithelial tissue layer (non-keratinized stratified squamous epithelium) of fast-growing and easily regenerated cells, kept moist with tears. The corneal epithelium is made up of epithelial cells and covers the front of the cornea. It acts as a frontline barrier to protect the cornea, resisting the free flow of fluids from the tears, and prevents bacteria from entering the cornea and inside of the eye.
Irregularity or defects of the corneal epithelium disrupts the smoothness of the air/tear-film interface, the most significant component of the total refractive power of the eye, thereby reducing visual acuity. It is continuous with the conjunctival epithelium, and is composed of about 6 layers of cells which are shed constantly on the exposed layer and are regenerated by multiplication in the basal layer. In dry eye disease, the corneal epithelium often becomes damaged.
In some methods, tear production is increased, or the thickness of the tear film coating the eye is increased. In some cases, tear retention is decreased, such that tears are evaporated quickly from the eye. Tearing (also known as lacrimation or lachrymation) is the reflex secretion of tears in response to external or internal irritants. Tears are a bodily fluid which may serve to clean and lubricate the eyes in response to irritation. In healthy mammalian eyes, the cornea is continually kept wet and nourished by basal tears. They lubricate the eye, and help to keep it clear of dust. Tear fluid contains water, mucin, lipids, lysozyme, lactoferrin, lipocalin, lacritin, immunoglobulins, glucose, urea, sodium, and potassium.
Some of the substances in lacrimal fluid (such as lysozyme) fight against bacterial infection as a part of the immune system. Lysozyme does this by dissolving a layer in the outer coating, called peptidoglycan, of certain bacteria. Tears are a typical body fluid with a salt content similar to blood plasma. Usually, in a 24-hour period, 0.75 to 1.1 grams (0.03-0.04 ounce avoirdupois) of tears is secreted; this rate slows with age. In addition, the basal tears are composed of antioxidants such as ascorbate, urate, cysteine, glutathione, and tyrosine. Ascorbate and urate constitute half of the tears.
A second type of tears results from irritation of the eye by foreign particles, or from the presence of irritant substances such as onion vapors, perfumes and other fragrances, tear gas, or pepper spray in the eye's environment, including the cornea, conjunctiva, or nasal mucosa, which trigger TRP channels in the ophthalmic nerve. It can also occur with bright light and hot or peppery stimuli to the tongue and mouth. It is also linked with vomiting, coughing and yawning. These reflex tears attempt to wash out irritants that may have come into contact with the eye.
The methods disclosed herein may relate to preservation or improvement of tear production, such as basal tears or reflex tears.
15 Patient selection In accordance with the methods of the invention, the methods may additionally comprise the step of selecting a subject for treatment with a therapeutically effective amount of a polypeptide comprising or consisting of LINK_TSG6.
The methods may comprise evaluating a subject or patient for evidence of, or susceptibility to, an ocular surface disorder such as dry eye disease, such as corneal damage (for example the presence of corneal epithelial lesions), inadequate tear production and inflammation (for example redness or swelling, or the presence of one or more biological markers of inflammation such as one or more of the pro-inflammatory cytokines TNFa, IL-113, IFN-y or IL-6 and MMP9).
The method may involve a comprehensive eye examination. This may include:
assessment of medical history to determine the patients signs and symptoms and to note any general health problems, medications or environmental factors that may be contributing to the dry eye problem; external examination of the eye, including lid structure and blink dynamics; evaluation of the eyelids and cornea using bright light and magnification; or measurement of the quantity and quality of tears for any abnormalities.
An individual may be determined to have a corneal epithelial defect, for example through staining with lissamine green, rose bengal or fluorescein dye.
The individual may be determined to have inadequate tear production, for example through using the phenol red thread test or Schirmer's test.
In some cases described herein, the individual to be treated has a condition that is associated with increased incidence of ocular surface disorders such as dry eye disease. In some cases the condition is an autoimmune condition. The autoimmune condition may be Sjogren's syndrome.
The individual may have been previously diagnosed as having Sjogren's syndrome. Sjogren's syndrome causes one of the most severe forms of dry eye disease, characterised by inflammatory destruction of lacrimal glands and the ocular surface. Inflammation of lacrimal glands and ocular surface is a key feature of dry eye disease, and plays an important role in the pathogenesis of dry eye disease and Sjogren's syndrome. In some cases, the autoimmune condition is rheumatoid arthritis or diabetes. In some cases, the individual has Type 1 diabetes or Type 2 diabetes.
In some cases, the ocular surface disorder such as dry eye disease does not involve inflammation. Dry eye disease not associated with inflammation may be associated with tear film instability and/or fast tear evaporation.
Treatments Disclosed herein are methods of treating or preventing ocular surface disorders such as dry eye disease.
The methods may involve the reduction or elimination of one or more signs and symptoms of an ocular surface disorder such as dry eye disease, such as a reduction in corneal epithelial defects or lesions, an increase in tear production, an increase or preservation of the number of goblet cells, a reduction in the
The methods may comprise evaluating a subject or patient for evidence of, or susceptibility to, an ocular surface disorder such as dry eye disease, such as corneal damage (for example the presence of corneal epithelial lesions), inadequate tear production and inflammation (for example redness or swelling, or the presence of one or more biological markers of inflammation such as one or more of the pro-inflammatory cytokines TNFa, IL-113, IFN-y or IL-6 and MMP9).
The method may involve a comprehensive eye examination. This may include:
assessment of medical history to determine the patients signs and symptoms and to note any general health problems, medications or environmental factors that may be contributing to the dry eye problem; external examination of the eye, including lid structure and blink dynamics; evaluation of the eyelids and cornea using bright light and magnification; or measurement of the quantity and quality of tears for any abnormalities.
An individual may be determined to have a corneal epithelial defect, for example through staining with lissamine green, rose bengal or fluorescein dye.
The individual may be determined to have inadequate tear production, for example through using the phenol red thread test or Schirmer's test.
In some cases described herein, the individual to be treated has a condition that is associated with increased incidence of ocular surface disorders such as dry eye disease. In some cases the condition is an autoimmune condition. The autoimmune condition may be Sjogren's syndrome.
The individual may have been previously diagnosed as having Sjogren's syndrome. Sjogren's syndrome causes one of the most severe forms of dry eye disease, characterised by inflammatory destruction of lacrimal glands and the ocular surface. Inflammation of lacrimal glands and ocular surface is a key feature of dry eye disease, and plays an important role in the pathogenesis of dry eye disease and Sjogren's syndrome. In some cases, the autoimmune condition is rheumatoid arthritis or diabetes. In some cases, the individual has Type 1 diabetes or Type 2 diabetes.
In some cases, the ocular surface disorder such as dry eye disease does not involve inflammation. Dry eye disease not associated with inflammation may be associated with tear film instability and/or fast tear evaporation.
Treatments Disclosed herein are methods of treating or preventing ocular surface disorders such as dry eye disease.
The methods may involve the reduction or elimination of one or more signs and symptoms of an ocular surface disorder such as dry eye disease, such as a reduction in corneal epithelial defects or lesions, an increase in tear production, an increase or preservation of the number of goblet cells, a reduction in the
16 expression of one or more inflammatory cytokines, or a reduction in redness, or burning sensations, pain or discomfort, or an improvement in visual signs and symptoms such as loss of vision or blurring of vision.
The methods may involve the treatment or prevention of corneal epithelial defects or lesions. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions as compared to the number and/or extent of corneal epithelial lesions prior to the treatment. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions as compared to the number and/or extent of corneal epithelial lesions in an untreated control, or a control treated with full length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The extent of corneal epithelial lesions may be reduced by at least 25%, at least 50%, at least 75%, or at least 100% compared to the control. The treatment may result in corneal epithelial lesions on 0%, less than 10%, less than 20%, less than 30%, less than 40% or less than 50% of the corneal surface.
The methods may involve the treatment or prevention of inadequate tear production. The methods may result in an increase in tear production, such as an increase in the volume of tear production. The methods may result in an increase in the volume of tears produced as compared to the volume of tears prior to the treatment. The methods may result in an increase in the volume of tears produced as compared to the volume of tears in an untreated control, or a control treated with full-length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The volume of tears may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more than 100% compared to the control.
The methods may involve increasing or preserving the number of goblet cells.
In this context, increasing or preserving the number of goblet cells means that the number of goblet cells after treatment with LINK_TSG6 is not decreased to the same extent as the decrease observed in the absence of the treatment, such as in an untreated control. In some cases, the number of goblet cells is increased or unchanged as compared to the number of goblet cells prior to the treatment.
The methods may involve a reduction in the levels of one or more inflammatory cytokines. For example, the method may result in a reduction in the levels of nucleic acids corresponding to one or more inflammatory cytokines. In some cases, the method results in a reduction in the levels inflammatory cytokine proteins. The inflammatory cytokines may be selected from TNFa, IL6, IFN-y, and IL-113. The levels may be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level before treatment, or as compared to an untreated control.
Treatment may result in complete resolution of the signs and symptoms of the ocular surface disorder such as dry eye disease.
Prevention may mean that no signs and symptoms of an ocular surface disorder such as dry eye disease emerge, or it may mean that the signs and symptoms of the ocular surface disorder such as dry eye disease develop to a lesser extent than in the absence of treatment.
The methods may involve the treatment or prevention of corneal epithelial defects or lesions. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions as compared to the number and/or extent of corneal epithelial lesions prior to the treatment. The methods may result in a decrease in the number and/or extent of corneal epithelial lesions as compared to the number and/or extent of corneal epithelial lesions in an untreated control, or a control treated with full length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The extent of corneal epithelial lesions may be reduced by at least 25%, at least 50%, at least 75%, or at least 100% compared to the control. The treatment may result in corneal epithelial lesions on 0%, less than 10%, less than 20%, less than 30%, less than 40% or less than 50% of the corneal surface.
The methods may involve the treatment or prevention of inadequate tear production. The methods may result in an increase in tear production, such as an increase in the volume of tear production. The methods may result in an increase in the volume of tears produced as compared to the volume of tears prior to the treatment. The methods may result in an increase in the volume of tears produced as compared to the volume of tears in an untreated control, or a control treated with full-length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The volume of tears may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more than 100% compared to the control.
The methods may involve increasing or preserving the number of goblet cells.
In this context, increasing or preserving the number of goblet cells means that the number of goblet cells after treatment with LINK_TSG6 is not decreased to the same extent as the decrease observed in the absence of the treatment, such as in an untreated control. In some cases, the number of goblet cells is increased or unchanged as compared to the number of goblet cells prior to the treatment.
The methods may involve a reduction in the levels of one or more inflammatory cytokines. For example, the method may result in a reduction in the levels of nucleic acids corresponding to one or more inflammatory cytokines. In some cases, the method results in a reduction in the levels inflammatory cytokine proteins. The inflammatory cytokines may be selected from TNFa, IL6, IFN-y, and IL-113. The levels may be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level before treatment, or as compared to an untreated control.
Treatment may result in complete resolution of the signs and symptoms of the ocular surface disorder such as dry eye disease.
Prevention may mean that no signs and symptoms of an ocular surface disorder such as dry eye disease emerge, or it may mean that the signs and symptoms of the ocular surface disorder such as dry eye disease develop to a lesser extent than in the absence of treatment.
17 Methods described herein may involve the topical administration of LINK_TSG6.
LINK_TSG6 may be topically administered to the eye (ocular delivery), preferably to the cornea, such as the surface of the cornea. In some cases, the LINK_TSG6 is administered as an eye drop. The LINK_TSG6 may be formulated as a topical liquid formulation, such as an eye drop. LINK_TSG6 may be formulated by suspension or emulsion.
Administration is preferably in a therapeutically effective amount. A
therapeutically effective amount of LINK_TSG6 may be determined according to various parameters, especially according to the polypeptide; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A therapeutically effective amount of LINK_TSG6 is an amount effective to ameliorate one or more signs and symptoms of an ocular surface disorder such as dry eye disease, such as to reduce the level of corneal damage or corneal lesions, increase lacrimal function, or increase lacrimal tear production, or reduce inflammation in the eye, such as inflammation in the cornea.
In some cases, LINK_TSG6 is administered at 12 pg-15 pg per eye, such as between 10 pg and 20 pg, or between 12 and 15 pg. In some cases, LINK_TSG6 is administered at about 8 pg per eye, about 9 pg per eye, about 10 pg per eye, about 11 pg per eye, about 12 pg per eye, about 13 pg per eye, about 14 pg per eye, or about 15 pg per eye.
In some cases, a higher dose is desired. In such cases, LINK_TSG6 is administered at about 120pg-150pg per eye. In some cases, the dosage of LINK_TSG6 is at least 80 pg, at least 90 pg, at least 100 pg, at least 110 pg, at least 120 pg, at least 130 pg, or at least 140 pg per eye. In some cases, the dosage of LINK_TSG6 is less than 170 pg, less than 160 pg, less than 150 pg, less than 140 pg, less than 130 pg or less than 120 pg per eye.
In some cases, an even higher dose is desired. For example, the dosage of LINK_TSG6 may be at least 170 pg, at least 180 pg, at least 190 pg, at least 200 pg, at least 210 pg at least 220 pg, at least 230 pg, at least 240 pg, at least 250 pg, at least 260 pg, at least 270 pg, at least 280 pg, at least 290 pg, at least 300 pg, at least 320 pg, at least 340 pg, at least 360 pg, at least 380 pg, least 400 pg, at least 420 pg, at least 440 pg, at least 460 pg, at least 480 pg, at least 500 pg, at least 550 pg, at least 600 pg, at least 650 pg, at least 700 pg, at least 750 pg, at least 800 pg, at least 900 pg, at least 1 mg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4mg, at least 1.5mg, at least 1.6mg or at least 1.7mg.
Administration may be once daily, twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, eight times daily, nine times daily, ten times daily or more than ten times daily.
Administration is preferably twice daily. In some preferred cases, the administration is more than twice daily, such as three or four times daily. In some preferred cases, the administration is less than four times daily, or less than three times daily, or two times daily or once daily.
In particularly preferred cases, administration is two times daily. In some cases, the treatment comprises administering LINK_TSG6 polypeptide once per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide less frequently than once per day, such as once every two days, one time every three days, once every week, or once every two weeks.
LINK_TSG6 may be topically administered to the eye (ocular delivery), preferably to the cornea, such as the surface of the cornea. In some cases, the LINK_TSG6 is administered as an eye drop. The LINK_TSG6 may be formulated as a topical liquid formulation, such as an eye drop. LINK_TSG6 may be formulated by suspension or emulsion.
Administration is preferably in a therapeutically effective amount. A
therapeutically effective amount of LINK_TSG6 may be determined according to various parameters, especially according to the polypeptide; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A therapeutically effective amount of LINK_TSG6 is an amount effective to ameliorate one or more signs and symptoms of an ocular surface disorder such as dry eye disease, such as to reduce the level of corneal damage or corneal lesions, increase lacrimal function, or increase lacrimal tear production, or reduce inflammation in the eye, such as inflammation in the cornea.
In some cases, LINK_TSG6 is administered at 12 pg-15 pg per eye, such as between 10 pg and 20 pg, or between 12 and 15 pg. In some cases, LINK_TSG6 is administered at about 8 pg per eye, about 9 pg per eye, about 10 pg per eye, about 11 pg per eye, about 12 pg per eye, about 13 pg per eye, about 14 pg per eye, or about 15 pg per eye.
In some cases, a higher dose is desired. In such cases, LINK_TSG6 is administered at about 120pg-150pg per eye. In some cases, the dosage of LINK_TSG6 is at least 80 pg, at least 90 pg, at least 100 pg, at least 110 pg, at least 120 pg, at least 130 pg, or at least 140 pg per eye. In some cases, the dosage of LINK_TSG6 is less than 170 pg, less than 160 pg, less than 150 pg, less than 140 pg, less than 130 pg or less than 120 pg per eye.
In some cases, an even higher dose is desired. For example, the dosage of LINK_TSG6 may be at least 170 pg, at least 180 pg, at least 190 pg, at least 200 pg, at least 210 pg at least 220 pg, at least 230 pg, at least 240 pg, at least 250 pg, at least 260 pg, at least 270 pg, at least 280 pg, at least 290 pg, at least 300 pg, at least 320 pg, at least 340 pg, at least 360 pg, at least 380 pg, least 400 pg, at least 420 pg, at least 440 pg, at least 460 pg, at least 480 pg, at least 500 pg, at least 550 pg, at least 600 pg, at least 650 pg, at least 700 pg, at least 750 pg, at least 800 pg, at least 900 pg, at least 1 mg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4mg, at least 1.5mg, at least 1.6mg or at least 1.7mg.
Administration may be once daily, twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, eight times daily, nine times daily, ten times daily or more than ten times daily.
Administration is preferably twice daily. In some preferred cases, the administration is more than twice daily, such as three or four times daily. In some preferred cases, the administration is less than four times daily, or less than three times daily, or two times daily or once daily.
In particularly preferred cases, administration is two times daily. In some cases, the treatment comprises administering LINK_TSG6 polypeptide once per day. In some cases, the treatment comprises administering LINK_TSG6 polypeptide less frequently than once per day, such as once every two days, one time every three days, once every week, or once every two weeks.
18 The dose, schedule, mode or time course of administration of the polypeptide of the invention can be modified according to response to therapy. For example, dose and/or the frequency of administration and/or time course of administration may be increased if response to therapy is suboptimal. Conversely, dose and/or the frequency of administration and/or time course of administration can be reduced if response to therapy is better than expected.
In some cases, the treatment involves the co-administration of LINK_TSG6 with artificial tears, prednisolone, cyclosporine, Lifitegrast (XiidraTm), or any combination thereof. Administration may be sequential or simultaneous. Preferably, where LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears, the administration is simultaneous or .. substantially simultaneous. Preferably, the co-administered agents are administered via the same route of administration, such as by topical administration to the eye.
Evaluation of a subject for an ocular surface disorder such as dry eye disease may occur at any point before, during or after administration of a therapeutically effective amount of LINK_TSG6. In some embodiments, the methods of the invention comprise commencing administration, evaluating a subject as .. above and based on the evaluation continuing, altering or discontinuing further administration. In some embodiments, altering administration comprises increasing or decreasing the dose and/or the frequency and/or the time course of administration.
Formulations Formulations suitable for ocular administration include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound. In preferred formulations, LINK_TSG6 is solubilised in a saline solution. In some formulations LINK_TSG6 is solubilised in PBS (phosphate buffered saline). As described herein, the inventors have discovered that LINK_TSG6 polypeptide has high solubility in saline as compared to full length TSG6. Described herein are compositions, particularly pharmaceutical compositions, comprising high concentrations of LINK_TSG6.
The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in saline. The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline. In some aspects, the pharmaceutical composition comprises at least 2000 pg/ml, 2100 pg/ml, .. 2200 pg/ml, 2300 pg/ml 2400pg/ml, 2500 pg/ml, 2600 pg/ml, 2700 pg/ml, 2800 pg/ml, 2900 pg/ml, 3000 pg/ml, 3100 pg/ml, 3200 pg/ml, 3300 pg/ml or more than 3300 pg/ml LINK_TSG6.
Preferably, the pharmaceutical formation comprises at least 2000 pg/ml LINK_TSG6.
Eye drop formulations as disclosed herein may further comprise one or more of a preservative, antioxidant, stabilizer, tonicity modifier, viscosity modifier or buffer.
Preferably, the eye drop formulation is .. a sterile eye drop formulation. In some cases, the eye drop formulation contains between about 240 pg/ml and about 300 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 12 pg and around 15pg of LINK_TSG6 per drop. Each drop may be around 50 pl. The eye drop formulation may
In some cases, the treatment involves the co-administration of LINK_TSG6 with artificial tears, prednisolone, cyclosporine, Lifitegrast (XiidraTm), or any combination thereof. Administration may be sequential or simultaneous. Preferably, where LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears, the administration is simultaneous or .. substantially simultaneous. Preferably, the co-administered agents are administered via the same route of administration, such as by topical administration to the eye.
Evaluation of a subject for an ocular surface disorder such as dry eye disease may occur at any point before, during or after administration of a therapeutically effective amount of LINK_TSG6. In some embodiments, the methods of the invention comprise commencing administration, evaluating a subject as .. above and based on the evaluation continuing, altering or discontinuing further administration. In some embodiments, altering administration comprises increasing or decreasing the dose and/or the frequency and/or the time course of administration.
Formulations Formulations suitable for ocular administration include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound. In preferred formulations, LINK_TSG6 is solubilised in a saline solution. In some formulations LINK_TSG6 is solubilised in PBS (phosphate buffered saline). As described herein, the inventors have discovered that LINK_TSG6 polypeptide has high solubility in saline as compared to full length TSG6. Described herein are compositions, particularly pharmaceutical compositions, comprising high concentrations of LINK_TSG6.
The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in saline. The pharmaceutical composition may comprise LINK_TSG6 polypeptide solubilised in phosphate buffered saline. In some aspects, the pharmaceutical composition comprises at least 2000 pg/ml, 2100 pg/ml, .. 2200 pg/ml, 2300 pg/ml 2400pg/ml, 2500 pg/ml, 2600 pg/ml, 2700 pg/ml, 2800 pg/ml, 2900 pg/ml, 3000 pg/ml, 3100 pg/ml, 3200 pg/ml, 3300 pg/ml or more than 3300 pg/ml LINK_TSG6.
Preferably, the pharmaceutical formation comprises at least 2000 pg/ml LINK_TSG6.
Eye drop formulations as disclosed herein may further comprise one or more of a preservative, antioxidant, stabilizer, tonicity modifier, viscosity modifier or buffer.
Preferably, the eye drop formulation is .. a sterile eye drop formulation. In some cases, the eye drop formulation contains between about 240 pg/ml and about 300 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 12 pg and around 15pg of LINK_TSG6 per drop. Each drop may be around 50 pl. The eye drop formulation may
19 comprise between 200 pg/ml and 350 pg/ml, between 200 pg/ml and 320 pg/ml, between 220 pg/ml and 320 pg/ml, or between 240 pg/ml and 300 pg/ml. Preferably, the eye drop formulation comprises between about 240 pg/ml and about 300 pg/ml. The eye drop formulation may contain at least 200 pg/ml, at least 220 pg/ml, at least 230 pg/ml, at least 240 pg/ml, at least 250 pg/ml, at least 260 pg/ml, at least 270 pg/ml, at least 280 pg/ml, at least 290 pg/ml, at least 300 pg/ml, at least 310 pg/ml, at least 320 pg/ml or more than 320 pg/ml LINK_TSG6 polypeptide.
In some cases, a higher dose formulation is desired. Such formulations may contain between 2400 and 3000 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 120 pg and around 150pg of LINK_TSG6 per drop. The eye drop formulation may comprise between 2000 pg/ml and 3500 pg/ml, between 2000 pg/ml and 3200 pg/ml, between 2200 pg/ml and 3200 pg/ml, or between 2400 pg/ml and 3000 pg/ml. Preferably, the eye drop formulation comprises between about 2400 pg/ml and about 3000 pg/ml. The eye drop formulation may contain at least 2000 pg/ml, at least 2200 pg/ml, at least 2300 pg/ml, at least 2400 pg/ml, at least 2500 pg/ml, at least 2600 pg/ml, at least 2700 pg/ml, at least 2800 pg/ml, at least 2900 pg/ml, at least 3000 pg/ml, at least 3100 pg/ml, at least 3200 pg/ml or more than 3200 pg/ml LINK_TSG6 polypeptide.
In the some cases, the eye drop formulation may further comprise artificial tears. Artificial tears are lubricant eye drops. Artificial tears may contain one or more agents selected from carboxymethyl cellulose, polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hyaluronan, water, salts and polymers, such as polyethylene glycol or polypropylene glycol.
In some cases, the eye drop formulation may further comprise prednisolone, cyclosporine, Lifitegrast (XiidraTM) or a combination of prednisolone and cyclosporine, prednisolone and Lifitegrast (XiidraTm), cyclosporine and Lifitegrast (XiidraTm), or prednisolone, cyclosporine and Lifitegrast (XiidraTm).
Pharmaceutical compositions may be prepared using a pharmaceutically acceptable "carrier" composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, loss, or change, of taste (ageusia) and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US
federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" refers to diluents, binders, lubricants and disintegrants.
Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.
.. The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability,
In some cases, a higher dose formulation is desired. Such formulations may contain between 2400 and 3000 pg/ml LINK_TSG6. Such formulations are useful for delivering between around 120 pg and around 150pg of LINK_TSG6 per drop. The eye drop formulation may comprise between 2000 pg/ml and 3500 pg/ml, between 2000 pg/ml and 3200 pg/ml, between 2200 pg/ml and 3200 pg/ml, or between 2400 pg/ml and 3000 pg/ml. Preferably, the eye drop formulation comprises between about 2400 pg/ml and about 3000 pg/ml. The eye drop formulation may contain at least 2000 pg/ml, at least 2200 pg/ml, at least 2300 pg/ml, at least 2400 pg/ml, at least 2500 pg/ml, at least 2600 pg/ml, at least 2700 pg/ml, at least 2800 pg/ml, at least 2900 pg/ml, at least 3000 pg/ml, at least 3100 pg/ml, at least 3200 pg/ml or more than 3200 pg/ml LINK_TSG6 polypeptide.
In the some cases, the eye drop formulation may further comprise artificial tears. Artificial tears are lubricant eye drops. Artificial tears may contain one or more agents selected from carboxymethyl cellulose, polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hyaluronan, water, salts and polymers, such as polyethylene glycol or polypropylene glycol.
In some cases, the eye drop formulation may further comprise prednisolone, cyclosporine, Lifitegrast (XiidraTM) or a combination of prednisolone and cyclosporine, prednisolone and Lifitegrast (XiidraTm), cyclosporine and Lifitegrast (XiidraTm), or prednisolone, cyclosporine and Lifitegrast (XiidraTm).
Pharmaceutical compositions may be prepared using a pharmaceutically acceptable "carrier" composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, loss, or change, of taste (ageusia) and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US
federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" refers to diluents, binders, lubricants and disintegrants.
Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.
.. The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability,
20 bioavailability, safety, and/or efficacy of the active ingredients, i.e.
LINK_TSG6 used in the composition.
Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives. Ointments are typically prepared from the active compound and a paraffinic or a water-miscible ointment base.
Creams are typically prepared from the active compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30%
w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the active compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus .. the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred .. esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to
LINK_TSG6 used in the composition.
Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives. Ointments are typically prepared from the active compound and a paraffinic or a water-miscible ointment base.
Creams are typically prepared from the active compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30%
w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the active compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus .. the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred .. esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to
21 be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.
For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of .. these theoretical explanations.
Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and "include", and variations such as "comprises", "comprising", and "including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an,"
and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means for example +/- 10%.
Examples EXAMPLE 1: LINK TSG6 is more soluble than full length TSG-6 The solubility and aggregation state of full-length TSG-6 and LINK_TSG6 in PBS
were compared using UV spectrophotometry and dynamic light scattering (DLS) at a range of concentrations. The LINK_TSG6 was fully soluble at 2 mg/ml and showed no aggregation at 0.4, 0.8, 1.6 or 3.2 mg/ml. The full-length protein had much lower solubility with less than 40% of the protein remaining in solution at 0.4, 0.8, 1.6.
and 3.2 mg/ml. The full-length protein was also highly aggregated at 0.2, 0.4.
0.8, 1.6 and 3.2 mg/ml. The DLS measurements were out of range for full-length TSG-6 at 1.6 and 3.2 mg/ml, indicating that aggregates of greater than 25 million Da were being formed.
EXAMPLE2: LINK TSG6 improves signs and symptoms of dry eye disease in a mouse model Topical TSG-6 has previously been shown to be as effective in inflammation mediated dry eye disease as cyclosporine eye drops (Kim et al., 2016). To test whether the LINK_TSG6 polypeptide was useful in this indication we investigated its effects in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes).
NOD.B10.H2b mice (12 week-old, Jackson Lab), a model for primary ocular Sjogren's syndrome (dry eye disease without diabetes) were treated with topically administered with LINK_TSG6 for 7 days. C57BL/6 mice were used as a negative control, as they do not develop spontaneous dry eye.
For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of .. these theoretical explanations.
Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and "include", and variations such as "comprises", "comprising", and "including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an,"
and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means for example +/- 10%.
Examples EXAMPLE 1: LINK TSG6 is more soluble than full length TSG-6 The solubility and aggregation state of full-length TSG-6 and LINK_TSG6 in PBS
were compared using UV spectrophotometry and dynamic light scattering (DLS) at a range of concentrations. The LINK_TSG6 was fully soluble at 2 mg/ml and showed no aggregation at 0.4, 0.8, 1.6 or 3.2 mg/ml. The full-length protein had much lower solubility with less than 40% of the protein remaining in solution at 0.4, 0.8, 1.6.
and 3.2 mg/ml. The full-length protein was also highly aggregated at 0.2, 0.4.
0.8, 1.6 and 3.2 mg/ml. The DLS measurements were out of range for full-length TSG-6 at 1.6 and 3.2 mg/ml, indicating that aggregates of greater than 25 million Da were being formed.
EXAMPLE2: LINK TSG6 improves signs and symptoms of dry eye disease in a mouse model Topical TSG-6 has previously been shown to be as effective in inflammation mediated dry eye disease as cyclosporine eye drops (Kim et al., 2016). To test whether the LINK_TSG6 polypeptide was useful in this indication we investigated its effects in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes).
NOD.B10.H2b mice (12 week-old, Jackson Lab), a model for primary ocular Sjogren's syndrome (dry eye disease without diabetes) were treated with topically administered with LINK_TSG6 for 7 days. C57BL/6 mice were used as a negative control, as they do not develop spontaneous dry eye.
22 Mice were treated by topical application of 1 pg LINK_TSG6, in 10 pl PBS, 4x per day (QID) for 7 days.
Mice were anesthetised with an intraperitoneal injection of zolazepam-tiletamine (Zoletil , Virbac, Carros, France), and either 10 pl LINK_TSG6 or PBS was administered using a pipette.
Mice were randomly assigned to treatment groups as follows:
1) Group 1 (Negative control): C57BL/6 mice, 12-week old (n=6, 12 eyes), untreated 2) Group 2 (Positive control): NOD.B10, 12-week old (n=6, 6 eyes) + PBS 10 pl QID
3) Group 3 (Experimental group): NOD.B10, 12-week old (n=8, 8 eyes) +
LINK_TSG6 1 pg (in 10 pl PBS) QID.
To assess the effect of treatment on dry eye disease, we undertook the following assays:
1) Corneal epithelial defects by vital staining (with lissamine green) of defect sites and scoring. After applying one drop of 3% lissamine Green B to the inferior lateral conjunctival sac of a mouse, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists as per the following ocular staining score system: score 0 for no punctuate staining; score 1 when less than one third of the corneal surface was stained; score 2 when two thirds or less was stained; and score 3 when more than two thirds was stained;
2) Lacrimal tear production examined by phenol red thread test;
3) Inflammatory cytokine expression by real time RT PCR in the cornea and intra-orbital gland; and 4) Conjunctival goblet cell count on PAS-stained conjunctival fornix. The number of PAS-stained cells was counted per 100 pm in four different sections of the eye from the same animal, and the average count was determined in each eye as the goblet cell density.
As shown in Figure 2A, after LINK_TSG6 treatment, corneal epithelial defects were significantly reduced (p < 0.01). There was a significant improvement in corneal epithelial defects between PBS- and Link_TSG6- treated groups (p < 0.001).
As shown in Figure 2B, tear production was significantly increased by LINK_TSG6 treatment (p < 0.01).
There was a significant improvement in tear production between PBS- and LINK_TSG6-treated groups (p <0.001).
Figure 2C shows that LINK_TSG6 treatment significantly suppressed mRNA levels of IFN-y, TNF-a and IL-113 in the cornea and intraorbital gland. LINK_TSG6-treated eyes had levels of cytokines similar to those of control C57BL/6 mice.
2D shows that LINK_TSG6 treatment significantly increased the number of conjunctival goblet cells (these cells produce the mucin components in tears).
Mice were anesthetised with an intraperitoneal injection of zolazepam-tiletamine (Zoletil , Virbac, Carros, France), and either 10 pl LINK_TSG6 or PBS was administered using a pipette.
Mice were randomly assigned to treatment groups as follows:
1) Group 1 (Negative control): C57BL/6 mice, 12-week old (n=6, 12 eyes), untreated 2) Group 2 (Positive control): NOD.B10, 12-week old (n=6, 6 eyes) + PBS 10 pl QID
3) Group 3 (Experimental group): NOD.B10, 12-week old (n=8, 8 eyes) +
LINK_TSG6 1 pg (in 10 pl PBS) QID.
To assess the effect of treatment on dry eye disease, we undertook the following assays:
1) Corneal epithelial defects by vital staining (with lissamine green) of defect sites and scoring. After applying one drop of 3% lissamine Green B to the inferior lateral conjunctival sac of a mouse, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists as per the following ocular staining score system: score 0 for no punctuate staining; score 1 when less than one third of the corneal surface was stained; score 2 when two thirds or less was stained; and score 3 when more than two thirds was stained;
2) Lacrimal tear production examined by phenol red thread test;
3) Inflammatory cytokine expression by real time RT PCR in the cornea and intra-orbital gland; and 4) Conjunctival goblet cell count on PAS-stained conjunctival fornix. The number of PAS-stained cells was counted per 100 pm in four different sections of the eye from the same animal, and the average count was determined in each eye as the goblet cell density.
As shown in Figure 2A, after LINK_TSG6 treatment, corneal epithelial defects were significantly reduced (p < 0.01). There was a significant improvement in corneal epithelial defects between PBS- and Link_TSG6- treated groups (p < 0.001).
As shown in Figure 2B, tear production was significantly increased by LINK_TSG6 treatment (p < 0.01).
There was a significant improvement in tear production between PBS- and LINK_TSG6-treated groups (p <0.001).
Figure 2C shows that LINK_TSG6 treatment significantly suppressed mRNA levels of IFN-y, TNF-a and IL-113 in the cornea and intraorbital gland. LINK_TSG6-treated eyes had levels of cytokines similar to those of control C57BL/6 mice.
2D shows that LINK_TSG6 treatment significantly increased the number of conjunctival goblet cells (these cells produce the mucin components in tears).
23 Overall, these results demonstrate that LINK_TSG6 significantly suppressed inflammation, reduced corneal epithelial defects, increased tear production, and increased conjunctival goblet cells in NOD.B10 dry eye mice.
EXAMPLE 3: Effects of LINK TSG6 on dry eye disease are dose-dependent We were interested to understand whether the effects observed in example 1 were dose dependent. To this end, we applied 1, 0.1, or 0.01 pg of LINK_TSG6, in 5 pl PBS, topically QID for 7 days to 12 week old NOD.B10.H2b mice (Jackson Lab).
Mice were randomly assigned to the following treatment groups:
1) Group 1 (Negative control): C57BL/6 mice, 12-week old (n=6, 12 eyes) untreated 2) Group 2 (Positive control): NOD.B10 (n=8, 8 eyes) + PBS 5 pl QID
3) Group 3 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 1 pg (in 5 pl PBS) QID
4) Group 4 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 0.1 pg QID
5) Group 5 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 0.01 pg QID
The effect of the treatment was determined as per example 1.
As shown in Figure 3A, corneal epithelial defects were quantified in lissamine green-stained corneas.
LINK_TSG6 1 pg and 0.1 pg was effective in reducing corneal epithelial defects, but LINK_TSG6 0.01 pg did not significantly improve corneal epithelial defects.
In Figure 3B, Phenol red threat test indicated that LINK_TSG6 1 pg and 0.1 pg was were effective in increasing the amount of tear production, but LINK_TSG6 0.01 pg did not significantly improve the amount of tear production.
As shown in Figures 3C and D, the highest dose of LINK_TSG6 tested, 1 pg was most effective in suppressing TNF-a expression.
Overall, these results demonstrate a dose-dependent improvement of dry eye parameters by topically applied LINK_TSG6 polypeptide (1 pg most effective and 0.01 pg least effective).
These effects were also observed with higher doses of LINK_TSG6. As shown in Figure 4A, the higher doses (1 pg and 10 pg) of LINK_TSG6 exhibited significantly less corneal epithelial damage as compared to PBS-treated mice. At the highest dose, many of the LINK_TSG6 treated eyes have no corneal lesions (score of 0), which is the same as the untreated negative control C57BL/6 mice. This shows that LINK_TSG6 can promote the healing or repair of epithelial lesions.
Dose dependent responses were also observed in respect of tear production (Figure 4B ¨ note that even the lowest dose of LINK_TSG6 tested has a significant effect on tear production), inflammation (Figure 4C) and conjunctival goblet cell numbers (Figure 4D).
EXAMPLE 3: Effects of LINK TSG6 on dry eye disease are dose-dependent We were interested to understand whether the effects observed in example 1 were dose dependent. To this end, we applied 1, 0.1, or 0.01 pg of LINK_TSG6, in 5 pl PBS, topically QID for 7 days to 12 week old NOD.B10.H2b mice (Jackson Lab).
Mice were randomly assigned to the following treatment groups:
1) Group 1 (Negative control): C57BL/6 mice, 12-week old (n=6, 12 eyes) untreated 2) Group 2 (Positive control): NOD.B10 (n=8, 8 eyes) + PBS 5 pl QID
3) Group 3 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 1 pg (in 5 pl PBS) QID
4) Group 4 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 0.1 pg QID
5) Group 5 (Experimental group): NOD.B10 (n=8, 8 eyes) + LINK_TSG6 0.01 pg QID
The effect of the treatment was determined as per example 1.
As shown in Figure 3A, corneal epithelial defects were quantified in lissamine green-stained corneas.
LINK_TSG6 1 pg and 0.1 pg was effective in reducing corneal epithelial defects, but LINK_TSG6 0.01 pg did not significantly improve corneal epithelial defects.
In Figure 3B, Phenol red threat test indicated that LINK_TSG6 1 pg and 0.1 pg was were effective in increasing the amount of tear production, but LINK_TSG6 0.01 pg did not significantly improve the amount of tear production.
As shown in Figures 3C and D, the highest dose of LINK_TSG6 tested, 1 pg was most effective in suppressing TNF-a expression.
Overall, these results demonstrate a dose-dependent improvement of dry eye parameters by topically applied LINK_TSG6 polypeptide (1 pg most effective and 0.01 pg least effective).
These effects were also observed with higher doses of LINK_TSG6. As shown in Figure 4A, the higher doses (1 pg and 10 pg) of LINK_TSG6 exhibited significantly less corneal epithelial damage as compared to PBS-treated mice. At the highest dose, many of the LINK_TSG6 treated eyes have no corneal lesions (score of 0), which is the same as the untreated negative control C57BL/6 mice. This shows that LINK_TSG6 can promote the healing or repair of epithelial lesions.
Dose dependent responses were also observed in respect of tear production (Figure 4B ¨ note that even the lowest dose of LINK_TSG6 tested has a significant effect on tear production), inflammation (Figure 4C) and conjunctival goblet cell numbers (Figure 4D).
24 EXAMPLE 4: Comparison of effects of full length TSG-6 protein and LINK TSG6 on dry eye disease Our experiments revealed that LINK_TSG6 polypeptide was effective at treating dry eye disease. We were interested to understand how this compared to full length (FL) TSG-6 (FL
TSG6). We compared equivalent molar doses of FL TSG6 and LINK_TSG6, administered 2x/day (BID).
12 week old NOD.B10 mice and C57BL/6 mice were randomly assigned to treatment groups as follows:
1) Group 1 NOD.B10 + PBS 5 pl BID for 7 days (positive control);
2) Group 2 NOD.B10 + full-length TSG-6 (FL TSG6) (R&D Systems) 5 p1(3.27, 0.327, 0.0327pg) BID for 7 days;
3) Group 3 NOD.B10 + LINK_TSG6 5 pl (1, 0.1, 0.01 pg) BID for 7 days; and 4) Group 4 C57BL/6 mice (negative control, no dry eye).
Dosages of 0.01, 0.1 and 1.0 micrograms LINK_TSG6 were chosen as per example 2. Mass spectrometry was used to determine the molecular weight of recombinant human TSG-6 (R&D Systems) as 35.7 kDa. Based on this determination, we calculated equimolar doses of 0.037, 0.37 and 3.7 micrograms for full-length TSG-6 (FL TSG6).
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
1) Lissamine green staining and score for corneal epithelial damage. After applying one drop of 3% Lissamine Green B to the inferior lateral conjunctival sac of a mouse, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists as per the following ocular staining score system: score 0 for no punctuate staining; score 0.5 for trace staining;
score 1 when less than one third of the corneal surface was stained; score 2 when two thirds or less was stained; and score 3 when more than two thirds was stained;
2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) As shown in Figure 5A, LINK_TSG6 resulted in a significant reduction in corneal epithelial lesions as compared to an equimolar concentration of full length TSG-6.
Figures 5B, 5C and 5D show that the highest dose tested, 1 pg LINK_TSG6, caused increased tear production, preserved goblet cells and reduced the number of CD3-stained inflammatory foci in the intraorbital gland, as compared to an equimolar dose (3.27 pg) of full length TSG-6.
TSG6). We compared equivalent molar doses of FL TSG6 and LINK_TSG6, administered 2x/day (BID).
12 week old NOD.B10 mice and C57BL/6 mice were randomly assigned to treatment groups as follows:
1) Group 1 NOD.B10 + PBS 5 pl BID for 7 days (positive control);
2) Group 2 NOD.B10 + full-length TSG-6 (FL TSG6) (R&D Systems) 5 p1(3.27, 0.327, 0.0327pg) BID for 7 days;
3) Group 3 NOD.B10 + LINK_TSG6 5 pl (1, 0.1, 0.01 pg) BID for 7 days; and 4) Group 4 C57BL/6 mice (negative control, no dry eye).
Dosages of 0.01, 0.1 and 1.0 micrograms LINK_TSG6 were chosen as per example 2. Mass spectrometry was used to determine the molecular weight of recombinant human TSG-6 (R&D Systems) as 35.7 kDa. Based on this determination, we calculated equimolar doses of 0.037, 0.37 and 3.7 micrograms for full-length TSG-6 (FL TSG6).
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
1) Lissamine green staining and score for corneal epithelial damage. After applying one drop of 3% Lissamine Green B to the inferior lateral conjunctival sac of a mouse, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists as per the following ocular staining score system: score 0 for no punctuate staining; score 0.5 for trace staining;
score 1 when less than one third of the corneal surface was stained; score 2 when two thirds or less was stained; and score 3 when more than two thirds was stained;
2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) As shown in Figure 5A, LINK_TSG6 resulted in a significant reduction in corneal epithelial lesions as compared to an equimolar concentration of full length TSG-6.
Figures 5B, 5C and 5D show that the highest dose tested, 1 pg LINK_TSG6, caused increased tear production, preserved goblet cells and reduced the number of CD3-stained inflammatory foci in the intraorbital gland, as compared to an equimolar dose (3.27 pg) of full length TSG-6.
25 EXAMPLE 5: Evaluation of LINK TSG6 in a desiccation injury-induced diy eye model and comparison with Restasis.
Having demonstrated the efficacy of LINK_TSG6 in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes), we were interested to understand whether LINK_TSG6 was also effective in treating individuals with the prevalent environmental evaporative dry eye disorder.
We use a desiccation model that better emulates an evaporative dry eye which is more prevalent form of dry eye, compared to the less prevalent Sjogren's syndrome-like dry eye. 7 week-old C57BL/6 mice were kept in a dry chamber and injected intraperitoneally with scopolamine three times daily for 10 days to induce desiccation injury. The air flow from an electric fan was allowed into the cage through the screen for 24 h, and humidity was maintained 30-35% inside the cage. Mice were divided into treatment groups as follows:
1) No desiccation injury (negative control, 2 mice, 4 eyes) 2) Group 1 (5 mice, 10 eyes): Desiccation injury + PBS 5 pl BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 0.1 pg (in PBS 5 pl) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 1 pg (in PBS 5 pl) BID for 10 days 5) Group 4 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 10 pg (in PBS 5 pl) BID for 10 days 6) Group 5 (5 mice, 10 eyes): Desiccation injury + Restasis (0.05%
cyclosporine A) 5 pL BID for 10 days After 10 days, signs and symptoms of dry eye were evaluated as follows;
1) Fluorescein staining and score for corneal epithelial damage (C57BL/6 mice are black and fluorescein stain is better for visualization than lissamine green) 2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) 4) Molecular assay of ocular surface and lacrimal glands (real time RT-PCR for inflammatory cytokines) Desiccation injury significantly induced corneal epithelial defects in C57BL/6 mice (p<0.01), as shown in Figure 6A (pre-desiccation and pre-treatment, showing animals selected for treatment had no corneal lesions at the start of the study) and 6B (post-desiccation and post-treatment), and LINK_TSG6 1 pg was effective at decreasing corneal epithelial defects under desiccation (p<0.001). Interestingly, Restasis did not significantly reduce corneal epithelial defects in this model, and LINK_TSG6 1 pg was better at decreasing corneal epithelial defects than Restasis (p<0.05).
Having demonstrated the efficacy of LINK_TSG6 in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes), we were interested to understand whether LINK_TSG6 was also effective in treating individuals with the prevalent environmental evaporative dry eye disorder.
We use a desiccation model that better emulates an evaporative dry eye which is more prevalent form of dry eye, compared to the less prevalent Sjogren's syndrome-like dry eye. 7 week-old C57BL/6 mice were kept in a dry chamber and injected intraperitoneally with scopolamine three times daily for 10 days to induce desiccation injury. The air flow from an electric fan was allowed into the cage through the screen for 24 h, and humidity was maintained 30-35% inside the cage. Mice were divided into treatment groups as follows:
1) No desiccation injury (negative control, 2 mice, 4 eyes) 2) Group 1 (5 mice, 10 eyes): Desiccation injury + PBS 5 pl BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 0.1 pg (in PBS 5 pl) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 1 pg (in PBS 5 pl) BID for 10 days 5) Group 4 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 10 pg (in PBS 5 pl) BID for 10 days 6) Group 5 (5 mice, 10 eyes): Desiccation injury + Restasis (0.05%
cyclosporine A) 5 pL BID for 10 days After 10 days, signs and symptoms of dry eye were evaluated as follows;
1) Fluorescein staining and score for corneal epithelial damage (C57BL/6 mice are black and fluorescein stain is better for visualization than lissamine green) 2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) 4) Molecular assay of ocular surface and lacrimal glands (real time RT-PCR for inflammatory cytokines) Desiccation injury significantly induced corneal epithelial defects in C57BL/6 mice (p<0.01), as shown in Figure 6A (pre-desiccation and pre-treatment, showing animals selected for treatment had no corneal lesions at the start of the study) and 6B (post-desiccation and post-treatment), and LINK_TSG6 1 pg was effective at decreasing corneal epithelial defects under desiccation (p<0.001). Interestingly, Restasis did not significantly reduce corneal epithelial defects in this model, and LINK_TSG6 1 pg was better at decreasing corneal epithelial defects than Restasis (p<0.05).
26 As shown in Figure 6C (pre-desiccation and pre-treatment) and 6D (post-desiccation and post-treatment), desiccation injury significantly reduced tear production in C57BL/6 mice (p<0.0001). As with the mouse model of primary ocular Sjogren's syndrome, LINK_TSG6 1 pg and 10 pg were effective at preserving tear production under desiccation (p<0.0001), and even 0.1 pg of LINK_TSG6 preserved tear production (p=0.0552). Although Restasis was effective at preserving tear production under desiccation (p<0.05), interestingly LINK_TSG6 1 pg was better at preserving tear production than Restasis (p<0.001).
It is well-known that Th1 and Th17 cells are increased in draining cervical lymph nodes (DLN) under desiccation stress, and that these cells are responsible for inducing dry eye.
We therefore performed FACS analysis on cervical draining lymph nodes to determine the level of Th1 (IFN-y+CD4+ cells) and Th17 cells (IL17A+CD4+ cells) in response to the various treatments. As shown in Figures 6E and 6F, LINK_TSG6 was also effective at suppressing Th1 and Th17 cells in cervical lymph nodes. Restasis had no such suppressive effect.
These data therefore indicate that LINK_TSG6 is also effective at treating dry eye disease, including in individuals with the prevalent environmental evaporative dry eye.
EXAMPLE 6: Evaluation of LINK TSG6 in established desiccation injury-induced diy eye Having demonstrated that LINK_TSG6 is effective at treating dry eye disease, including in individuals with the prevalent environmental evaporative dry eye, we were interested to understand whether LINK_TSG6 was also effective in treating individuals with established dry eye, to reverse the effects of the disease.
We used the same desiccation model as in Example 5, this time allowing the dry eye disease to become established for one week prior to treatment with LINK_TSG6. LINK_TSG6 was applied to the ocular surface after desiccation injury had been exerted to the ocular surface. The mice were then kept in the dry chamber with scopolamine injections for the 10 further days during which the treatments were administered.
7 week-old C57BL/6 mice were kept in a dry chamber and injected intraperitoneally with scopolamine three times daily for 7 days to establish dry eye. Mice were divided into treatment groups as follows:
1) No desiccation injury (negative control, 2 mice, 4 eyes) 2) Group 1(5 mice, 10 eyes): Desiccation injury + PBS 5 pl BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 0.1 pg (in PBS 5 pl) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 1 pg (in PBS 5 pl) BID for 10 days 5) Group 4 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 10 pg (in PBS 5 pl) BID for 10 days.
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
It is well-known that Th1 and Th17 cells are increased in draining cervical lymph nodes (DLN) under desiccation stress, and that these cells are responsible for inducing dry eye.
We therefore performed FACS analysis on cervical draining lymph nodes to determine the level of Th1 (IFN-y+CD4+ cells) and Th17 cells (IL17A+CD4+ cells) in response to the various treatments. As shown in Figures 6E and 6F, LINK_TSG6 was also effective at suppressing Th1 and Th17 cells in cervical lymph nodes. Restasis had no such suppressive effect.
These data therefore indicate that LINK_TSG6 is also effective at treating dry eye disease, including in individuals with the prevalent environmental evaporative dry eye.
EXAMPLE 6: Evaluation of LINK TSG6 in established desiccation injury-induced diy eye Having demonstrated that LINK_TSG6 is effective at treating dry eye disease, including in individuals with the prevalent environmental evaporative dry eye, we were interested to understand whether LINK_TSG6 was also effective in treating individuals with established dry eye, to reverse the effects of the disease.
We used the same desiccation model as in Example 5, this time allowing the dry eye disease to become established for one week prior to treatment with LINK_TSG6. LINK_TSG6 was applied to the ocular surface after desiccation injury had been exerted to the ocular surface. The mice were then kept in the dry chamber with scopolamine injections for the 10 further days during which the treatments were administered.
7 week-old C57BL/6 mice were kept in a dry chamber and injected intraperitoneally with scopolamine three times daily for 7 days to establish dry eye. Mice were divided into treatment groups as follows:
1) No desiccation injury (negative control, 2 mice, 4 eyes) 2) Group 1(5 mice, 10 eyes): Desiccation injury + PBS 5 pl BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 0.1 pg (in PBS 5 pl) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 1 pg (in PBS 5 pl) BID for 10 days 5) Group 4 (5 mice, 10 eyes): Desiccation injury + LINK_TSG6 10 pg (in PBS 5 pl) BID for 10 days.
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
27 1) Fluorescein staining and score for corneal epithelial damage (B6 mice are black and fluorescein stain is better for visualization than lissamine green);
2) Phenol red thread test for lacrimal tear secretion; and 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining).
As shown in Figure 7A LINK_TSG6 was effective at decreasing corneal epithelial defects at all concentrations tested. As shown in Figure 7B, at all concentrations tested, LINK_TSG6 was effective at increasing aqueous tear production. Significant changes were observed even for the lowest concentration tested.
Figure 7C demonstrates that 1 pg and 10 pg of LINK_TSG6 resulted in an increase in goblet cell number as compared to control, restoring to levels comparable to those observed in the absence of desiccation.
Moreover, as shown in Figure 7D, treatment with LINK_TSG6 was effective at suppressing the level of MMP-9 mRNA in the ocular surface.
These data therefore indicate that LINK_TSG6 is effective at treating established dry eye disease, in addition to reducing the severity of desiccation injury. These data support the use of LINK_TSG6 for the treatment of individuals with dry eye disorder, including in individuals with the prevalent environmental evaporative dry eye.
EXAMPLE 7: Evaluation of LINK TSG6 in a dry eye model and comparison with Restasis.
Having demonstrated the superiority of LINK_TSG6 to Restasis in treating evaporative dry eye, we were interested to compare LINK_TSG6 to Restasis in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes), as used in Example 4.
12 week old NOD.B10 mice and C57BL/6 mice were randomly assigned to treatment groups as follows:
1) Group 1 C57BL/6 mice (negative control, no dry eye/desiccation).
2) Group 2 NOD.B10 + PBS 5 pl BID for 7 days (positive control);
3) Group 3 NOD.B10 + LINK_TSG6 5 pl (0.1, 1, 10 pg/5 pl) BID for 7 days; and 4) Group 4 NOD.B10 + Restasis 5 pl BID for 7 days;
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
1) Lissamine green staining and score for corneal epithelial damage.
2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) As shown in Figure 8B and D, and in agreement with our studies in Example 4, LINK_TSG6 resulted in a significant reduction in corneal epithelial lesions and increase in tear production in the NOD.B10 mouse
2) Phenol red thread test for lacrimal tear secretion; and 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining).
As shown in Figure 7A LINK_TSG6 was effective at decreasing corneal epithelial defects at all concentrations tested. As shown in Figure 7B, at all concentrations tested, LINK_TSG6 was effective at increasing aqueous tear production. Significant changes were observed even for the lowest concentration tested.
Figure 7C demonstrates that 1 pg and 10 pg of LINK_TSG6 resulted in an increase in goblet cell number as compared to control, restoring to levels comparable to those observed in the absence of desiccation.
Moreover, as shown in Figure 7D, treatment with LINK_TSG6 was effective at suppressing the level of MMP-9 mRNA in the ocular surface.
These data therefore indicate that LINK_TSG6 is effective at treating established dry eye disease, in addition to reducing the severity of desiccation injury. These data support the use of LINK_TSG6 for the treatment of individuals with dry eye disorder, including in individuals with the prevalent environmental evaporative dry eye.
EXAMPLE 7: Evaluation of LINK TSG6 in a dry eye model and comparison with Restasis.
Having demonstrated the superiority of LINK_TSG6 to Restasis in treating evaporative dry eye, we were interested to compare LINK_TSG6 to Restasis in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes), as used in Example 4.
12 week old NOD.B10 mice and C57BL/6 mice were randomly assigned to treatment groups as follows:
1) Group 1 C57BL/6 mice (negative control, no dry eye/desiccation).
2) Group 2 NOD.B10 + PBS 5 pl BID for 7 days (positive control);
3) Group 3 NOD.B10 + LINK_TSG6 5 pl (0.1, 1, 10 pg/5 pl) BID for 7 days; and 4) Group 4 NOD.B10 + Restasis 5 pl BID for 7 days;
Signs and symptoms of dry eye disease were determined as per the previous examples, namely:
1) Lissamine green staining and score for corneal epithelial damage.
2) Phenol red thread test for lacrimal tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) As shown in Figure 8B and D, and in agreement with our studies in Example 4, LINK_TSG6 resulted in a significant reduction in corneal epithelial lesions and increase in tear production in the NOD.B10 mouse
28 model of primary ocular Sjogren's syndrome. Moreover, LINK_TSG6 promoted healing to a greater extent than Restasis at both the 1 and 10 pg doses, resulting in reduced corneal lesions, preservation in the number of goblet cells, and reduced number of CD3-stained inflammatory foci in the intraorbital gland, as compared to Restasis. This shows that even at the lowest doses tested, LINK_TSG6 was better at treating dry eye in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes) than Restasis, as previously observed in preventing the development of, or treating established, evaporative dry eye disease. These data further support the use of LINK_TSG6 for the treatment of individuals with dry eye disorder.
Claims (17)
1. LINK_TSG6 polypeptide for use in the treatment or prevention of an ocular surface disorder such as dry eye disease.
2. The LINK_TSG6 polypeptide for use according to claim 1, wherein the treatment or prevention of an ocular surface disorder such as dry eye disease comprises one or more effects selected from the group consisting:
healing of corneal epithelial defects;
increase in tear production;
suppression of inflammation; and a higher number of conjunctival goblet cells.
healing of corneal epithelial defects;
increase in tear production;
suppression of inflammation; and a higher number of conjunctival goblet cells.
3. The LINK_TSG6 polypeptide for use according to claim 1 or claim 2 wherein the treatment comprises topical administration of LINK_TSG6 polypeptide to the eye.
4. The LINK_TSG6 polypeptide for use according to any one of claims 1 to 3 wherein the treatment comprises administering LINK_TSG6 polypeptide two times per day.
5. The LINK_TSG6 polypeptide for use according to any one of claims 1 to 3 wherein the treatment comprises administering LINK_TSG6 polypeptide less than four times per day.
6. The LINK_TSG6 polypeptide for use according to claim 3 wherein the LINK_TSG6 polypeptide is formulated as an eye drop.
7. The LINK_TSG6 polypeptide for use according to any one of the preceding claims wherein LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears.
8. The LINK_TSG6 polypeptide for use according to any one of the preceding claims wherein about 10pg-200pg LINK_TSG6 polypeptide is administered per eye, preferably 120pg-150pg LINK_TSG6 polypeptide is administered per eye.
9. The LINK_TSG6 polypeptide for use according to claim 2, wherein the suppression of inflammation comprise a decrease in the production of one or more pro-inflammatory cytokines in the cornea or the intraorbital lacrimal glands, the pro-inflammatory cytokines optionally selected from TNF-a, IL-6, IFN-y and IL-18.
10. The LINK_TSG6 polypeptide for use according to claim 2 wherein the treatment or prevention of dry eye disease comprises healing of corneal epithelial defects, increase in tear production, suppression of inflammation or increase in number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells prior to the administration of LINK_TSG6 polypeptide.
11. The LINK_TSG6 polypeptide for use according to claim 2 wherein the treatment or prevention of dry eye disease comprises healing of corneal epithelial defects, increase in tear production, suppression of inflammation or increase in number of conjunctival goblet cells as compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells as compared to a control individual treated with full-length TSG-6 protein.
12. The LINK_TSG6 polypeptide for use according to any one of the preceding claims wherein the individual being treated has Sjögren's syndrome.
13. The LINK_TSG6 polypeptide for use according to any one of the preceding claims wherein the treatment comprises the administration of an eye drop comprising LINK_TSG6.
14. The LINK_TSG6 polypeptide for use according to any one of the preceding claims, wherein the LINK_TSG6 polypeptide comprises, consist, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9.
15. An eye drop formulation comprising LINK_TSG6 polypeptide.
16. The eye drop formulation according to claim 15 comprising between 240pg/ml and 3000pg/ml, preferably between 2400pg/ml and 3000pg/ml.
17. The eye drop formulation according to claim 15 or claim 16 further comprising prednisolone, cyclosporine, Lifitegrast (XiidraTM) or artificial tears.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB1910645.9A GB201910645D0 (en) | 2019-07-25 | 2019-07-25 | Treatment for dry eye disease |
GB1910645.9 | 2019-07-25 | ||
PCT/EP2020/067448 WO2021013452A1 (en) | 2019-07-25 | 2020-06-23 | Tsg6 polypeptide fragment for dry eye disease |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3148714A1 true CA3148714A1 (en) | 2021-01-28 |
Family
ID=67990534
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3148714A Pending CA3148714A1 (en) | 2019-07-25 | 2020-06-23 | Tsg6 polypeptide fragment for dry eye disease |
Country Status (8)
Country | Link |
---|---|
US (1) | US20220288161A1 (en) |
EP (1) | EP4003396A1 (en) |
JP (1) | JP2022542889A (en) |
KR (1) | KR20220047979A (en) |
CN (1) | CN114555107A (en) |
CA (1) | CA3148714A1 (en) |
GB (1) | GB201910645D0 (en) |
WO (1) | WO2021013452A1 (en) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9878003B2 (en) | 2006-03-06 | 2018-01-30 | The University Of Manchester | Method of treating bone disorders using TSG-6 |
US8785395B2 (en) | 2010-05-03 | 2014-07-22 | The Texas A & M University System | Adult stem cells/progenitor cells and stem cell proteins for treatment of eye injuries and diseases |
US20160075750A1 (en) | 2011-11-04 | 2016-03-17 | Darwin J. Prockop | Production of TSG-6 Protein |
-
2019
- 2019-07-25 GB GBGB1910645.9A patent/GB201910645D0/en not_active Ceased
-
2020
- 2020-06-23 EP EP20734889.7A patent/EP4003396A1/en active Pending
- 2020-06-23 KR KR1020227004454A patent/KR20220047979A/en unknown
- 2020-06-23 WO PCT/EP2020/067448 patent/WO2021013452A1/en active Application Filing
- 2020-06-23 CA CA3148714A patent/CA3148714A1/en active Pending
- 2020-06-23 US US17/597,802 patent/US20220288161A1/en active Pending
- 2020-06-23 JP JP2022504600A patent/JP2022542889A/en active Pending
- 2020-06-23 CN CN202080053382.8A patent/CN114555107A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JP2022542889A (en) | 2022-10-07 |
US20220288161A1 (en) | 2022-09-15 |
GB201910645D0 (en) | 2019-09-11 |
KR20220047979A (en) | 2022-04-19 |
CN114555107A (en) | 2022-05-27 |
WO2021013452A1 (en) | 2021-01-28 |
EP4003396A1 (en) | 2022-06-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Inamoto et al. | Ocular graft-versus-host disease after hematopoietic cell transplantation: Expert review from the Late Effects and Quality of Life Working Committee of the CIBMTR and Transplant Complications Working Party of the EBMT | |
Vijmasi et al. | Topical administration of lacritin is a novel therapy for aqueous-deficient dry eye disease | |
Utine et al. | Clinical review: topical ophthalmic use of cyclosporin A | |
US10881693B2 (en) | Processes for making and using a mesenchymal stem cell derived secretome | |
Hwang et al. | Comparison of clinical efficacies of autologous serum eye drops in patients with primary and secondary Sjögren syndrome | |
Ambroziak et al. | Immunomodulation on the ocular surface: a review | |
KR20230169375A (en) | Compositions comprising pedf-derived short peptides and uses thereof | |
US20210299219A1 (en) | Compositions and methods for prevention and treatment of corneal haze and scarring | |
Oh et al. | Effect of hypotonic 0.18% sodium hyaluronate eyedrops on inflammation of the ocular surface in experimental dry eye | |
Li et al. | Comparison of 0.3% hypotonic and isotonic sodium hyaluronate eye drops in the treatment of experimental dry eye | |
Jin et al. | Anti‑inflammatory effects of glycine thymosin β4 eye drops in experimental dry eye | |
CA3060331A1 (en) | Coversin for the treatment of cicatrising eye inflammatory disorders | |
EP3096777B1 (en) | Goblet cell replacement therapy | |
JP2023521086A (en) | Freeze-dried mesenchymal stem cell-derived secretome and use thereof | |
US20220288161A1 (en) | Tsg6 polypeptide fragment for dry eye disease | |
Chiang et al. | The ocular graft-versus-host disease: the path from current knowledge to future managements | |
JP6335188B2 (en) | Protein SLURP-1 for use in the treatment of eye diseases | |
Datta et al. | TRPA1 and TPRV1 ion channels are required for contact lens-induced corneal parainflammation and can modulate levels of resident corneal immune cells | |
Lee et al. | Superior limbic keratoconjunctivitis: Update on pathophysiology and management | |
CN114206849A (en) | Therapeutic methods for treating non-infectious ocular immunoinflammatory disorders | |
RU2021107211A (en) | OPHTHALMIC COMPOSITIONS FOR THE TREATMENT OF DRY EYE SYNDROME | |
JP2024504642A (en) | Treatment of xerophthalmia using Tampanacept ophthalmic composition | |
WO2013148155A1 (en) | Compositions and methods for the treatment of dry eye disease | |
JP2019525923A (en) | How to treat dry eye syndrome |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20240514 |